Ribosomal DNA restriction analysis reveals genetic heterogeneity in Saccharomyces cerevisiae Meyen ex Hansen.

A ribosomal DNA restriction analysis of 17 strains belonging to the Saccharomyces cerevisiae complex revealed two major groups, one of which corresponded to Saccharomyces bayanus. Our results generally correlate with previously reported genetic and molecular data and support the conclusion that S. bayanus should be reinstated as a separate taxon.     

The inhibition of cell division in Saccharomyces cerevisiae (Meyen) by carbon dioxide

A specific inhibiting effect of CO² on cell division in Saccharomycescerevisiae (Meyen) was shown. Two strains of S. cerevisiae weregrown in chemically-defined media in specially-designed pressurechambers equipped with sensitive pressure-measuring devices.The chambers were pressurized with 40 psi of N₂ or CO₂. Inhibitionof cell division and of production of new buds was not causedby N₂ but was caused by CO₂ when either endogenously producedor added. In contrast, metabolic production of CO₂ was unaffectedby endogenously-produced pressures which totally inhibited celldivision. Bud formation and new-cell formation (cell division) were almosttotally inhibited by 40 psi of added CO₂ when compared withaerated cultures. The DNA content per cell, however, was nearlytwice as great in the CO₂-treated cultures as in the controls.Thus inhibition of cell division in S. cerevisiae must occurby some mechanism other than by inhibition of DNA replication. (Received January 5, 1971; )     

The genus Saccharomyces (Meyen) Reess

The speciesSaccharomyces heterogenicus Osterwalder,Saccharomyces steineri Lodder et Kreger-van Rij,Saccharomyces pastorianus Hansen,Saccharomyces bayanus Saccardo andSaccharomyces willianus Saccardo were compared. It was found that the biochemical characters used for the definition of these species were variable and the strains of the given species were therefore evaluated together. With reference to the present accepted criteria of species specificity, grouping on the basis of coefficients of similarity was not conclusive. One of the characters (the fermentation and assimilation of galactose) was therefore dispensed with, while a new characteristic (determination) of amylolytic activity was added. It was found that the speciesSaccharomyces pastorianus, Saccharomyces bayanus andSaccharomyces willianus were so similar that they could be grouped in one species.Saccharomyces heterogenicus retained its species specific characteristics, whileSaccharomyces steineri formed a transition between this species and the other three. The coefficients of similarity, S, calculated by the methods of Jaccard and of Sokal and Michener, were compared and some of the more problematical questions of the numerical method are discussed.     

RNA-dependent ATPase from Saccharomyces cerevisiae

A new RNA-dependent ATPase has been isolated from yeast chromatin extracts and partially characterized. The protein has a sedimentation coefficient of about 7 S. The enzyme hydrolyzes specifically ATP (or dATP) to ADP (or dADP) and Pi in the presence of Mg2+ or Mn2+ ions and requires a single-stranded polynucleotide as cofactor. The order of efficiency of synthetic polymers is poly(rU) > poly(rI) greater than or equal to poly(dU) > poly(rA) greater than or equal to poly(rC). Among natural polymers, single-stranded DNA and poly(rA)-containing mRNA from yeast are also active but less so than poly(rU). The enzyme exhibits a pH optimum of 8 and is fully inhibited by 0.25 M NaCl. The Km for ATP is0.2 mM. The resemblance between this ATPase and DNA-dependent ATPases from other sources, as well as the termination factor rho, is discussed.     

Actin from Saccharomyces cerevisiae.

Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth.     

Elution of exocellular enzymes from Saccharomyces fragilis and Saccharomyces cerevisiae

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Elution of exocellular enzymes from Saccharomyces fragilis and Saccharomyces cerevisiae. J. Bacteriol. 91:1-13. 1966.-Invertase and acid phosphatase are repressible exocellular enzymes in Saccharomyces fragilis and S. cerevisiae. The conditions for eluting these enzymes from both organisms were compared. Either KCl or beta-mercaptoethanol eluted the enzymes from S. fragilis, and the amounts eluted varied quantitatively according to the physiological age of the organism. In addition to eluting enzymatic activity from the cells, these reagents also caused a large increase in the amount of activity that remained associated with the cells of S. fragilis. Invertase and acid phosphatase were not removed from cells of S. cerevisiae by KCl or beta-mercaptoethanol. These enzymes were separated from S. cerevisiae cells only when there was some degree of cell-wall digestion by snail gut fluid.     

Classical NLS proteins from Saccharomyces cerevisiae

Proteins can enter the nucleus through various receptor-mediated import pathways. One class of import cargos carries a classical nuclear localization signal (cNLS) containing a short cluster of basic residues. This pathway involves importin α (Impα), which possesses the cNLS binding site, and importin β (Impβ), which translocates the import complex through the nuclear pore complex. The defining criteria for a cNLS protein from Saccharomyces cerevisiae are an in vivo import defect in Impα and Impβ mutants, direct binding to purified Impα, and stimulation of this binding by Impβ. We show for the first time that endogenous S. cerevisiae proteins Prp20, Cdc6, Swi5, Cdc45, and Clb2 fulfill all of these criteria identifying them as authentic yeast cNLS cargos. Furthermore, we found that the targeting signal of Prp20 is a bipartite cNLS and that of Cdc6 is a monopartite cNLS. Basic residues present within these motifs are of different significance for the interaction with Impα. We determined the binding constants for import complexes containing the five cNLS proteins by surface plasmon resonance spectrometry. The dissociation constants for cNLS/α/β complexes differ considerably, ranging from 1 nM for Cdc6 to 112 nM for Swi5, suggesting that the nuclear import kinetics is determined by the strength of cNLS/Impα binding. Impβ enhances the affinity of Impα for cNLSs approximately 100-fold. This stimulation of cNLS binding to Impα results from a faster association in the presence of Impβ, whereas the dissociation rate is unaffected by Impβ. This implies that, after entry into the nucleus, the release of Impβ by the Ran guanosine triphosphatase (Ran GTPase) from the import complex is not sufficient to dissociate the cNLS/Impα subcomplex. Our observation that the nucleoporin Nup2, which had been previously shown to release the cNLS from Impα in vitro, is required for efficient import of all the genuine cNLS cargos supports a general role of Nup2 in import termination.     

Apurinic endonucleases from Saccharomyces cerevisiae.

Three endonuclease activities have been partially purified from Saccharomyces cerevisiae on the basis of their activity against x-irradiated closed-circular supercoiled bacteriophage PM2 DNA. These endonucleases also nick apurinic DNA and two out of the three activities incise DNA UV-irradiated with high doses. The endonuclease activities have also been distinguished on the basis of their magnesium requirement and sensitivity to EDTA.     

Membrane-associated phosphatidylinositol kinase from Saccharomyces cerevisiae

Membrane-associated phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was partially purified 93-fold from Saccharomyces cerevisiae. Activity was dependent on magnesium ions (10 mM) and the optimum pH was 8.5. The apparent Km values for ATP and phosphatidylinositol were 0.21 mM and 71 microM, respectively. Activity was stimulated by sodium cholate and inhibited by sodium, potassium, lithium, and fluoride ions.     

Apurinic endonuclease from Saccharomyces cerevisiae.

An endonuclease cleaving depurinated and alkylated double-stranded DNA has been purified 500-fold from Saccharomyces cerevisiae, strain MB 1052. The enzyme has an Mr of 31 000 +/- 2000, a sedimentation value of 3.2S and a diffusion coefficient of 9.5 X 10-7 cm2/s. The enzyme was active only at apurinic/apyridiminic sites, regardless of whether they were produced by heating the DNA at acidic pH or by alkylation with the ultimate carcinogen methyl methanesulphonate. Native DNA was not acted upon. U.v.-irradiated DNA and DNA treated with the ultimate carcinogen N-acetoxy-2-acetylaminofluorene were cleaved to an extent related to the extent of apurinic/apyridiminic sites. Enzymic activity was not dependent upon Mg2+, but was stimulated approx. 3-fold by 4mM-Mg2+. The enzyme did not bind to DEAE-cellulose or CM-cellulose at KCl concentrations greater than 160 mM. The endonuclease was obtained free of exonuclease and 3-methyladenine-DNA glycosylase activity in five chromatographic steps.     

Saccharomyces cerevisiae

Yeast prions are infectious proteins that spread exclusively by mating. The frequency of prions in the wild therefore largely reflects the rate of spread by mating counterbalanced by prion growth slowing effects in the host. We recently showed that the frequency of outcross mating is about 1% of mitotic doublings with 23–46% of total matings being outcrosses. These findings imply that even the mildest forms of the [PSI+], [URE3] and [PIN+] prions impart > 1% growth/survival detriment on their hosts. Our estimate of outcrossing suggests that Saccharomyces cerevisiae is far more sexual than previously thought and would therefore be more responsive to the adaptive effects of natural selection compared with a strictly asexual yeast. Further, given its large effective population size, a growth/survival detriment of > 1% for yeast prions should strongly select against prion-infected strains in wild populations of Saccharomyces cerevisiae.     

Natural hybrids from Saccharomyces cerevisiae, Saccharomyces bayanus and Saccharomyces kudriavzevii in wine fermentations

Several wine isolates of Saccharomyces were analysed for six molecular markers, five nuclear and one mitochondrial, and new natural interspecific hybrids were identified. The molecular characterization of these Saccharomyces hybrids was performed based on the restriction analysis of five nuclear genes (CAT8, CYR1, GSY1, MET6 and OPY1, located in different chromosomes), the ribosomal region encompassing the 5.8S rRNA gene and the two internal transcribed spacers, and sequence analysis of the mitochondrial gene COX2. This method allowed us to identify and characterize new hybrids between Saccharomyces cerevisiae and Saccharomyces kudriavzevii, between S. cerevisiae and Saccharomyces bayanus, as well as a triple hybrid S. bayanusxS. cerevisiaexS. kudriavzevii. This is the first time that S. cerevisiaexS. kudriavzevii hybrids have been described which have been involved in wine fermentation.