Restriction endonuclease analysis of ribosomal DNA from Saccharomyces cerevisiae

Summary Temperature-sensitive mutants of Escherichia coli that are unable to grow at high temperature can be obtained among those selected for resistance to streptovaricin or rifampicin at low temperature (Yura et al., 1970). One of these mutants (KY5323) that was supposed to carry a single mutation affecting both rifampicin resistance and temperature sensitivity was further investigated. Using purified RNA polymerase preparations obtained from the mutant and the wild type, it was found that the activity for DNA chain elongation is more sensitive to heat treatment than that for RNA chain initiation or DNA binding, and that the mutant enzyme is significantly more labile than the wild-type enzyme with respect to RNA chain elongation, when heat treatment is carried out at high salt concentration. These results, taken together with those of the enzyme reconstitution experiments, strongly suggest that the subunit of the polymerase is directly involved in both RNA chain initiation and elongation reactions. Enzyme reconstitution experiments using isolated subunits derived from the mutant and the wild-type polymerases demonstrate that the alteration of subunit is primarily responsible for both rifampicin resistance and thermolability of the mutant enzyme. In addition, the results suggested the apparent alteration of both and subunits in this mutant. Extensive transduction experiments provided genetic evidence that are consistent with the view that the strain KY5323 carries a second mutation affecting the subunit, beside the primary mutation affecting the subunit. The hypothetical subunit mutation seems to modify quantitatively the rifampicin resistance caused by the subunit mutation.     

Saccharomyces cerevisiae mutants defective in the maturation of ribosomal RNA

Summary Slow-growing mutants were isolated after mutagenesis of the osmotic-sensitive strain Saccharomyces cerevisiae VY1160. The isolated mutants in rich media have generation times from 300 to 400 min at 30°C. Studies on the biosynthesis of rRNA^x have shown, that the processing of 37S pre-rRNA in 6 of the slow-growing mutants occurs 3 to 4 times slower than in the parental strain. These mutants with decreased rate of rRNA maturation are of two different types. In some of them the processing of both 37S and 27S pre-rRNA is slowed down, while the mutants from the second group are acharacterized by a specific inhibition of the step 27S pre-rRNA25S rRNA. Experiments in which the synthesis of macromolecules was studied, have shown that in the mutants and in the parental strain, RNA and proteins are synthesized at comparable rates. Preliminary results suggest that the decreased rate of rRNA processing in three of the isolated mutants might be due to an insufficient function of the enzymes involved in the maturation of rRNA.Abbreviations rRNA ribosomal RNA - pre-rRNA precursor to ribosomal RNA     

The Saccharomyces cerevisiae homologue of ribosomal protein S26

The nucleotide sequence of RPS26, the gene encoding a homologue of ribosomal protein small subunit S26 in Saccharomyces cerevisiae, was determined. The deduced amino-acid sequence showed significant identity with its counter- parts from Neurospora crassa, human, rat and Arabidopsis thaliana. Disruption of RPS26 resulted in the formation of micro-colonies, suggesting that it is important for the normal cell growth of S. cerevisiae.     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

 We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome. Received: 9 February 1996/Accepted: 7 July 1996     

Calcium and Saccharomyces cerevisiae

The claim that Ca may be a dispensable element for yeast Saccharomyces cerevisiae has been reexamined. The cells of S. cerevisiae could grow in media which contained no added Ca and were deprived of contaminating Ca²⁺ by filtration through a Chelex 100 column. Also, the cells were able to grow in the presence of fairly high concentrations of EGTA. The apparent intracellular concentrations of Ca, assessed from the content of radioactive ⁴⁵Ca in cells preloaded with ⁴⁵CaCl₂, could vary within the range of approx. 2 nM to 2.8 mM, without adversively affecting growth or morphology of the cells. An extremely low affinity for Ca²⁺ of the system taking up Ca into the cells was corroborated. However, even the Chelex 100-treated media were found in contain 1–5 μM Ca when maintained in glass culture vessels. Also, the ability of the cells to take up Ca from a medium containing surplus of EGTA or EDTA was demonstrated. su14CEDTA, alone or in the presence of Ca, could also be transported into the cells. It has been inferred that Ca must be as essential for yeast as it is for other eucaryotic organisms. The omnipresence of contaminating Ca and peculiarities of the Ca transporting system, combined with an intricate intracellular compartmentation of Ca, would account for the impossibility to prove the importance of Ca for yeast by direct growth studies.     

Histones from Saccharomyces cerevisiae

Total histones and histone fractions isolated from Saccharomyces cerevisiae chromatin were analysed by polyacrylamide gel electrophoresis. The presence of the four histone fractions H2a, H2b, H3 and H4 was demonstrated. In addition, yeast chromatin contained a protein similar to histone H1 from mammals in molecular weight, charge and association properties with Triton X-100. However, it had a much lower lysine to arginine ratio, equal to about 3, as compared with H1 histones from higher eukaryotes. The order of electrophoretic mobilities of yeast histone fractions in acidic urea-polyacrylamide gels was similar to that observed for histones from plant sources, i.e. H4>H3>H2a>H2b>H1. Previously undetected protein (protein X) was extracted from yeast chromatin with 5 % HClO₄. The properties of this protein are under investigation.     

Mitochondrial DNA synthesis during the cell cycle of Saccharomyces cerevisiae

Logarithmically growing cultures of Saccharomyces cerevisiae were separated into fractions of increasing average age by zonal centrifugation. Nuclear and mitochondrial DNA labeled for both long and short periods were examined at different times in the cell cycle following isolation of the DNA on CsCl gradients. The data thus obtained were used to determine the relative times of replication. In S. cerevisiae the synthesis of nuclear and mitochondrial DNA was found to occur at the same time in the cell cycle. Some data indicate that mitochondrial DNA synthesis was completed before nuclear DNA synthesis.     

Petite-negative mutants of Saccharomyces cerevisiae

Summary A series of yeast mutants has been isolated with the inability to grow on fermentable carbon sources whilst growing normally on ethanol media. One of the mutants, namely MC16/206 lacks pyruvate decarboxylase activity and does not grow on glucose at 37°C but grows on both ethanol and glucose at 27°C. In this strain rho ^- petites are non-viable.     

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

Summary We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 1100 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictivetemperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress. Received: 17 April 1996 / Accepted: 21 October 1996     

Prezygotic reproductive isolation between Saccharomyces cerevisiae and Saccharomyces paradoxus

Background  

Matings between different Saccharomyces sensu stricto yeast species produce sexually sterile hybrids, so individuals should avoid mating with other species. Any mechanism that reduces the frequency of interspecific matings will confer a selective advantage. Here we test the ability of two closely-related Saccharomyces sensu stricto species to select their own species as mates and avoid hybridisation.     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

Timing of mitochondrial DNA synthesis during meiosis in Saccharomyces cerevisiae

Mitochondrial DNA (mtDNA) synthesis was examined during meiosis in Saccharomyces cerevisiae using an aneuploid strain disomic (n + 1) for chromosome III. The aneuploid has the advantage over true diploid strains in that it completes early meiotic events, including premeiotic chromosome replication, but does not form mature ascospores. Thus, differential extraction problems, resulting from the simultaneous presence of both unsporulated cells and spores in the population, are eliminated. The kinetic of mtDNA synthesis was monitored by determining the actual mtDNA content of cells following analytical CsCl centrifugation of cell extracts. MtDNA synthesis started soon after the cells were placed in sporulation medium and continued at an approximately constant rate until 24 h, resulting in slightly more than a doubling of the mitochondrial DNA content per cell. [¹⁴C]uracil was incorporated into mtDNA during the entire developmental period. Extensive preferential labeling of mtDNA occurred between 24 and 50 h, when no net DNA synthesis was observed.     

Mitotic gene conversion of large DNA heterologies in Saccharomyces cerevisiae

Summary Gene conversion of large DNA heterologous fragments has been shown to take place efficiently in Saccharomyces cerevisiae. It has been found that a 2.6 kb LEU2 DNA fragment in a multicopy plasmid was replaced by a 3.1 kb PG11 chromosomal DNA fragment, when both fragments were flanked by homologous DNA regions. Gene conversion was asymmetric in a total of 481 recombinants analyzed. In contrast, truncated PG11 or LEU2 genes in multicopy plasmids, gave no recombinants that restored a complete plasmid copy of these genes in a total of 242 recombinants studied, confirming that a conversion tract is disrupted by a heterologous region. The asymmetry of the events detected suggest that gene conversion of large DNA heterologies involves a process whereby a gap first covers one heterologous fragment and then this is followed by new DNA synthesis using the other heterologous fragment as a template. Therefore, it is likely that large DNA heterologies are converted by a double-strand gap repair mechanism.     

Analysis of rho mutability in Saccharomyces cerevisiae

Summary Two additional types of nuclear determinants involved in the control of spontaneous mutability of rho in S. cerevisiae have been identified: mmc and the pet-ts 1, 2, 10, 52 and 53 genes.These genes in their mutated recessive form increase at various extents the number of respiratory deficient cytoplasmic petite mutants accumulated.The gene mmc does not affect the respiratory activity and is not temperature-dependent whereas the pet-ts genes determine at the non permissive temperature a respiratory deficient phenotypes even if they affect the mutability of rho at the permissve and at the non permissive temperature.The data here reported suggest that a replicative complex exists for the mitochondrial DNA.It is in the purpose of this paper to deal with the relative contrition that mmc and pet-ts gene products have in ensuring the fidelity of this replicative complex.     

Recessive nonsense-suppression in yeast Saccharomyces cerevisiae

Summary The shift of recessive suppressor mutant of yeast Saccharomyces cerevisiae from permissive to restrictive conditions is accompanied by polysome decay and accumulation of 80 S ribosomes (Smirnov et al., 1976). In this paper some properties of 80 S ribosomes are studied. It is demonstrated that polysome decay under non-permissive conditions is not the consequence of the impairement of RNA synthesis. More than 70% of 80 S ribosomes accumulated under non-permissive conditions contain bound peptidyl-tRNAs localized in P-ribosomal site. tRNA moiety of bound peptidyl-tRNA is able to accept all 20 natural amino acids after chemical deacylation. Therefore it is not a specific isoacceptor species but rather total tRNA that is bound to ribosomes. The polypeptide residues of these peptidyl-tRNAs are heterogeneous in size. Their molecular weights are comparable with the molecular weights of the completed polypeptides. Some of the 80 S ribosomes accumulated under non-permissive conditions contain poly-A RNA. In conclusion, possible mechanism of the impairement of translation under non-permissive conditions in recessive suppressor strain is discussed.     

Plasmid stability in recombinant Saccharomyces cerevisiae

Because of many advantages, the yeast Saccharomyces cerevisiae is increasingly being employed for expression of recombinant proteins. Usually, hybrid plasmids (shuttle vectors) are employed as carriers to introduce the foreign DNA into the yeast host. Unfortunately, the transformed host often suffers from some kind of instability, tending to lose or alter the foreign plasmid. Construction of stable plasmids, and maintenance of stable expression during extended culture, are some of the major challenges facing commercial production of recombinant proteins. This review examines the factors that affect plasmid stability at the gene, cell, and engineering levels. Strategies for overcoming plasmid loss, and the models for predicting plasmid instability, are discussed. The focus is on S. cerevisiae, but where relevant, examples from the better studied Escherichia coli system are discussed. Compared to free suspension culture, immobilization of cells is particularly effective in improving plasmid retention, hence, immobilized systems are examined in some detail. Immobilized cell systems combine high cell concentrations with enhanced productivity of the recombinant product, thereby offering a potentially attractive production method, particularly when nonselective media are used. Understanding of the stabilizing mechanisms is a prerequisite to any substantial commercial exploitation and improvement of immobilized cell systems.