Copper uptake by whole cells and protoplasts of a wild-type and copper-resistant strain of Saccharomyces cerevisiae

Abstract A stable copper-resistant mutant of Saccharomyces cerevisiae took up less copper than the wild-type. The use of protoplasts showed that the decreased uptake depended on changed membrane transport properties and not on alterations in the cell wall.     

Osmotic adjustment and the accumulation of organic solutes in whole cells and protoplasts of Saccharomyces cerevisiae

In the presence of a suitable carbon source, whole cells and protoplasts of Saccharomyces cerevisiae synthesized glycerol as a compatible organic solute in response to increased external osmotic pressure. Boyle-van't Hoff plots showed that protoplasts, and non-turgid cells, exhibited a linear relationship between volume and the external osmotic pressure (i.e. they behaved as near-ideal osmometers), and that both protoplasts and cells have a component which is not osmotically responsive--the non-osmotic volume (NOV). Glycerol levels in whole cells and protoplasts were elevated by increased external osmotic pressure over a similar time-scale to the period of exponential cell growth, reaching a maximum value at 6-12 h and declining thereafter. This suggests that the restoration of turgor pressure in whole cells was not the sole regulator of glycerol accumulation. Stationary phase whole cells had negligible levels of intracellular glycerol after growth in a medium of raised osmotic pressure. However, intracellular trehalose synthesis in these cells began earlier and reached a higher maximum level than in basal medium. Once exponential growth had stopped, cell turgor and internal osmotic pressure decreased somewhat. These new, lower values may be determined by the extent of trehalose accumulation in stationary phase cells.     

Uptake of nystatin by protoplasts of sensitive and resistant cells of Saccharomyces cerevisiae

The uptake of nystatin by protoplasts derived from sensitive and resistant cells of Saccharomyces cerevisiae has been studied as a function of nystatin concentration, temperature and pH. The presence or absence of glucose in the uptake experiments was also studied. Activation energies (Ea) for nystatin uptake revealed profound differences between protoplasts derived from sensitive and resistant cells. Those for the latter closely resembled their whole cell counterparts. The values of Ea for the uptake of nystatin under all the conditions studied indicate the importance of the cell wall in the uptake process.     

Production of mead by immobilized whole cells of Saccharomyces cerevisiae

Summary The continuous production of mead was achieved with whole cells of Saccharomyces cerevisiae immobilized in calcium alginate gels. The alcohol production was stable in the pH range of 2.5–6.0 and a temperature range of 18–30°C with a sharp increase at 35°C. The process reduced the problems of contamination and secondary fermentation which are associated with traditional mead production.     

Scanning electron microscopy of cells and protoplasts ofSaccharomyces rouxii

Cells ofSaccharomyces rouxii from a normal broth culture were subjected to a high osmotic pressure (2 M KCl), fixed in 3% glutaraldehyde fortified with 2 M KCl, and then processed routinely for examination in a scanning electron microscope. Micrographs revealed birth and bud scars typical for the genus and an apparently undamaged surface topography. Protoplasts were prepared from the same material by digestion of cell walls with snail gut enzymes in the presence of 2 M KCl. Naked protoplasts were obtained and these exhibited surface invaginations. In addition, spheroidal protrusions were noted and these structures were equated with the periplasmic bodies previously described by transmission electron microscopy. The propensity for periplasmic body formation inSaccharomyces rouxii is contrasted with otherSaccharomyces species and the circumstantial evidence that relates periplasmic bodies to cryptic β-fructofuranosidase inS. rouxii is briefly discussed.     

Isolation and characterization of triacylglycerol-secreting mutant strain from yeast,Saccharomyces cerevisiae

To establish the molecular bases for development of a microbiological system approaching excretive fermentation of useful lipids, a mutant strain that accumulates lipids in the medium was isolated from the laboratory yeast Saccharomyces cerevisiae. Following the mutagenesis to strain YP1, a long chain fatty acid utilizer with ethylmethane sulfonate, the mutant strain, STG1, was selected from about 80,000 colonies. The analysis of extracellular lipids and the monitoring of leakage of intracellular proteins indicated that strain STG1 secreted lipids containing triacylglycerols into the extracellular space without cell lysis. Genetic studies clarified that this mutation was recessive and was complemented by wild-type genomic DNA fragments. STG1 was considered to be a good tool for elucidation of the molecular mechanism for transmembrane lipid transport.     

Phosphofructokinase-1 from Saccharomyces cerevisiae: analysis of molecular structure and function by electron microscopy and self-catalysed affinity labelling

Conventional and cryoelectron microscopy portray native octameric yeast phosphofructokinase-1 (PFK) as consisting of two identical heterotetrameric tetrahedron-like moieties being rotated relative to each other. Immunoelectron microscopy employing subunit-specific IgG identifies alpha-type subunits in the contact zone of the two tetrahedrons, while beta-chains are recognized exclusively at the tips of the octamer. The chemical reaction of phosphofructokinase with analogues of fructose 6-phosphate followed by autocatalytic phosphoryl transfer from [gamma-32P]-ATP results in a specific labelling of the alpha-subunit. AMP and fructose 2,6-bisphosphate affect labelling by stimulating the binding of substrate analogue; AMP additionally promotes phosphoryl transfer. No stimulation of labelling is observed with proteolytically modified tetrameric 12-S phosphofructokinase.     

The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37 degrees C, whereas the same cells did not grow at 23 degrees C. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23 degrees C but not at 37 degrees C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterization of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three-dimensional structure. The potential utility of these mutants is discussed.     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

High-voltage electron microscopy of whole,critical-point dried plant cells

Summary The lower epidermis ofSelaginella Helvetica leaves has numerous chloroplasts. In the diffuse light of the plant's normal habitat these are distributed over the inner wall of the cell, while in bright sunlight they move to the lateral walls. High voltage electron microscopy of whole critical-point dried cells shows that in the diffuse-light position the chloroplasts are connected by bundles of tightly-packed parallel filaments; these are distinct from, but seem to interconnect with, the filaments of the cytomatrix. In thin sections these appear as conventional microfilament bundles, while staining with rhodamineconjugated phalloidin implies that they are composed of actin. In bright light, when the chloroplasts have moved to the lateral walls, these microfilament bundles completely disappear, while filaments of the cytomatrix system remain attached to the chloroplasts. These results suggest that the function of the microfilament bundles may be to anchor the chloroplasts as much as to move them, and that the cytomatrix system may play a part in the movement; it is possible that actin microfilament bundles may actually dissociate into separate filaments within the cytomatrix. Staining of cryo-sections with FITC-labelled antitubulin reveals a typical cortical pattern of microtubules which appears to play no part in chloroplast motility.Abbreviations EDTA ethylenediaminetetra-acetic acid - EM electron microscopy - FITC fluorescein-iso-thiocyanate - HVEM high voltage electron microscopy - PIPES piperazine-NN-bis-2-ethanesulphonic acid     

The isolation, characterization and nucleotide sequence of the phosphoglucoisomerase gene of Saccharomyces cerevisiae

We have isolated the gene which encodes the glycolytic enzyme phosphoglucoisomerase (PGI) from the yeast Saccharomyces cerevisiae by functional complementation of a yeast mutant deficient in PGI activity with DNA from a wild-type yeast genomic library. The cloned gene has been localized by hybridization of specific DNA fragments to total yeast poly(A)+ RNA and by complementation of the mutant phenotype with subclones. The gene is expressed as an abundant mRNA of 1.9-kb and encodes a protein of 554 amino acids with an Mr of 61310. The nucleotide sequence of the gene as well as the 5' and 3' flanking regions are presented. The predicted PGI amino acid sequence shows a high degree of homology with the sequence predicted for human and mouse neuroleukin, a putative neurotropic factor. The codon usage within the coding region is very restricted, characteristic of a highly expressed yeast gene.     

The RAD3 gene of Saccharomyces cerevisiae: isolation and characterization of a temperature-sensitive mutant in the essential function and of extragenic suppressors of this mutant

Summary Mutations in the RAD3 gene of Saccharomyces cerevisiae were generated by integration of a mutagenized incomplete copy of the cloned gene into wild-type cells. Integrants were mass screened for colonies with abnormal growth characteristics at 37°C. A single temperature-sensitive mutant (rad3ts-1) was isolated and was shown to result from a missense mutation at codon 73 of the RAD3 gene. When shifted from 30° C to 37° C the strain undergoes only 2–4 cell doublings. This phenotype can be rescued by plasmids in which the essential function of the cloned RAD3 gene is intact, but not plasmids in which this function is inactivated. The mutant strain is weakly sensitive to ultraviolet (UV) radiation at restrictive temperatures. Measurement of RNA, DNA and protein synthesis at various times after shifting to restrictive temperatures does not show preferential inactivation of any one of these parameters and the temperature-sensitive mutation does not cause arrest at any specific phase of the cell cycle. The rad3ts-1 strain was transformed with multicopy plasmids from a normal yeast genomic library and two plasmids that partially suppress the temperature-sensitive phenotype were isolated. These suppressor genes (designated SRE1 and SRE2) are distinct from RAD3 and do not suppress the phenotype of several other temperature-sensitive mutants tested. Mutant strains carrying disruptions of the SRE1 gene are viable and are not sensitive to UV or radiation.     

The REC41 gene of Saccharomyces cerevisiae: isolation and genetic analysis

Recombination-deficient strains have been proven useful for the understanding of the genetic control of homologous recombination. As the genetic screens used to isolate recombination-deficient (rec(-)) yeast mutants have not been saturated, we sought to develop a simple colony color assay to identify mutants with low or elevated rates of recombination. Using this system we isolated a collection of rec(-) mutants. We report the characterization of the REC41 gene identified in this way. REC41 is required for normal levels of interplasmid recombination and gamma-ray induced mitotic interchromosomal recombination. The rec41-1 mutant failed to grow at 37 degrees C. Microscopic analysis of plated cells showed that 45-50% of them did not form visible colonies at permissive temperature. Haploid cells of the rec41 mutant show the same gamma-ray sensitivity as wild type ones. However, the diploid rec41 mutant shows gamma-ray sensitivity which is comparable with heterozygous REC41/rec41-1 diploid cells. This fact indicates semidominance of the rec41-1 mutation. Diploid strains homozygous for the rec41 rad52 mutations had the same gamma-ray sensitivity as single rad52 diploids and exhibited dramatically decreased growth rate. The expression of the HO gene does not lead to inviability of rec41 cells. The rec41 mutation has an effect on meiosis, likely meiotic recombination, even in the heterozygous state. We cloned the REC41 gene. Sequence analysis revealed that the REC41 gene is encoded by ORF YDR245w. Earlier, this ORF was attributed to MNN10, BED1, SLC2, CAX5 genes. Two multicopy plasmids with suppressers of the rec41-1 mutation (pm21 and pm32) were isolated. The deletion analysis showed that only DNA fragments with the CDC43 and HAC1 genes can partially complement the rec41-1 mutation.     

Isolation and characterization of an actinomycin D-sensitive mutant of Saccharomyces cerevisiae.

A single mutation in Saccharomyces cerevisiae conferred sensitivity to low concentrations of actinomycin D. Treatment with actinomycin D preferentially inhibited synthesis of rRNA's. Residual rRNA synthesized was processed normally. Total protein synthesis and inducibility of the enzyme maltase were relatively unaffected at concentrations of actinomycin D which severely inhibited rRNA synthesis.