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  • Articles  (82)
  • Chemical Engineering
  • Electronic structure and strongly correlated systems
  • Saccharomyces cerevisiae
  • Springer  (82)
  • 2000-2004  (18)
  • 1980-1984  (64)
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  • Articles  (82)
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  • 1
    ISSN: 1572-8773
    Keywords: major facilitator superfamily ; iron transport ; siderophores ; enterobactin ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3Δ background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using 55Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.
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  • 2
    ISSN: 1432-0983
    Keywords: Key words Citrinin ; Pet mutants ; Mitochondrial biogenesis ; Vacuolar ATPase ; YKL118W disruption ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In countries with a hot climate the mycotoxin citrinin represents a serious problem in fungal food-poisoning. In humans the renal system is affected the most and the mitochondrial respiratory chain was identified as a possible sensitive target for this toxin. In addition, citrinin has an antifungal activity that also inhibits the growth of the yeast Saccharomyces cerevisiae. So far the precise mode of action and the subcellular targets for citrinin have not been identified. Therefore, we decided to use the model organism yeast for a genetic approach to identify genes that play a role in the sensitivity against this mycotoxin. A large collection of conditional respiratory deficient yeast mutants was screened for sensitivity against citrinin. One special pet-ts mutant was identified that exhibited a higher sensitivity against citrinin. The genetic system of yeast allowed the isolation of the respective wild-type gene. This yeast gene encodes the Vph2p subunit that is essential for the correct assembly of the vacuolar ATPase. Isolation of the mutated gene and gene-disruption experiments of VPH2 and the partially overlapping small YKL118W gene verified this finding. The wild-type VPH2 gene restores all defects of the mutants. In contrast to this, YKL118W gave no complementation and the null mutant showed no phenotype. Thereby the yeast vacuolar ATPase was found to be important for the toxic effect of citrinin in yeast cells. The consequences of this finding for the molecular mechanism of citrinin action and its relation to the mitochondrial respiratory chain are discussed.
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  • 3
    ISSN: 1432-0983
    Keywords: Key wordsPOL32 ; SRS2 ; DNA repair ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pol32 is a subunit of Saccharomyces cerevisiae DNA polymerase δ required in DNA replication and repair. To gain insight into the function of Pol32 and to determine in which repair pathway POL32 may be involved, we extended the analysis of the pol32Δ mutant with respect to UV and methylation sensitivity, UV-induced mutagenesis; and we performed an epistasis analysis of UV sensitivity by combining the pol32Δ with mutations in several genes for postreplication repair (RAD6 group), nucleotide excision repair (RAD3 group) and recombinational repair (RAD52 group). These studies showed that pol32Δ is deficient in UV-induced mutagenesis and place POL32 in the error-prone RAD6/REV3 pathway. We also found that the increase in the CAN1 spontaneous forward mutation of different rad mutators relies entirely or partially on a functional POL32 gene. Moreover, in a two-hybrid screen, we observed that Pol32 interacts with Srs2, a DNA helicase required for DNA replication and mutagenesis. Simultaneous deletion of POL32 and SRS2 dramatically decreases cellular viability at 15 °C and greatly increases cellular sensitivity to hydroxyurea at the permissive temperature. Based on these findings, we propose that POL32 defines a link between the DNA polymerase and helicase activities, and plays a role in the mutagenic bypass repair pathway.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 38 (2000), S. 264-270 
    ISSN: 1432-0983
    Keywords: Key words Endopolygalacturonase ; Saccharomyces cerevisiae ; Kluyveromyces marxianus ; Pectinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The gene encoding endopolygalacturonase (EC 3.2.1.15) has been cloned, sequenced and expressed from three strains of Saccharomyces cerevisiae (including non-secretors) and three strains of Kluyveromyces marxianus. Both control and coding regions showed small differences within each species, one including loss of a potential glycosylation site. Two non-secreting S. cerevisiae strains (FY1679 and var. uvarum) had non-transcribed copies of functional genes. Maximum enzyme activity was achieved with the S. cerevisiae FY1679 gene in an expressing vector, with an enzyme activity of 51 μmol of reducing sugar released from polygalacturonic acid μg protein−1 min−1, the highest so far reported for a yeast.
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  • 5
    ISSN: 1432-0983
    Keywords: Key words Translation release factors ; Chromosome stability ; Microtubules ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chromosome stability in suppressor mutants for SUP35 and SUP45 genes coding for translation release factors was studied. We obtained spontaneous and UV-induced sup35 or sup45 mutants in a haploid strain disomic for chromosome III and tested the stability of an extra copy of this chromosome. The majority of the mutants showed increased chromosome instability. This phenotype was correlated with an increased sensitivity to the microtubule-poisoning drug benomyl which affects chromosome segregation at anaphase. Our data suggest that termination-translation factors eRF3 and eRF1 control chromosome transmission at mitotic anaphase in Saccharomyces cerevisiae.
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  • 6
    Electronic Resource
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    Springer
    Journal of thermal analysis and calorimetry 59 (2000), S. 643-648 
    ISSN: 1572-8943
    Keywords: drying ; intracellular water ; Saccharomyces cerevisiae ; TG
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The intracellular water content of a microorganism is an important parameter which is a determinant factor of its physiological properties. It is usually measured by complex and time consuming procedures. Thermogravimetry using infrared balance has been used for this purpose, through the identification of different drying steps occurring during the analysis. This work employs the same method with much smaller samples, using conventional thermogravimetric equipment in a simpler and faster way than other conventional procedures. Commercial yeast (Saccharomyces cerevisiae ) washed samples are analyzed in isothermal procedures which are run in about 30 min. The drying rate curve, when plotted as a function of the residual mass of the cells, allows the identification of the step where the intracellular water is lost and the determination of its content. The obtained values, on extracellular water free basis, are in the range of 65 to 69% and agree with those measured by other techniques.
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  • 7
    ISSN: 1573-4943
    Keywords: Homology modeling ; rotational energy barrier ; simulated annealing ; pyridoxal 5′-diphosphoadenosine ; pyridoxal 5′-triphosphoadenosine ; Saccharomyces cerevisiae ; phosphoenolpyruvate carboxykinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Molecular mechanics calculations have been employed to obtain models of the complexes between Saccharomyces cerevisiae phosphoenolpyruvate (PEP) kinase and the ATP analogs pyridoxal 5′-diphosphoadenosine (PLP-AMP) and pyridoxal 5′-triphosphoadenosine (PLP-ADP), using the crystalline coordinates of the ATP-pyruvate-Mn2+-Mg2+ complex of Escherichia coli PEP carboxykinase [Tari et al. (1997), Nature Struct. Biol. 4, 990–994]. In these models, the preferred conformation of the pyridoxyl moiety of PLP-ADP and PLP-AMP was established through rotational barrier and simulated annealing procedures. Distances from the carbonyl-C of each analog to ε-N of active-site lysyl residues were calculated for the most stable enzyme-analog complex conformation, and it was found that the closest ε-N is that from Lys290, thus predicting Schiff base formation between the corresponding carbonyl and amino groups. This prediction was experimentally verified through chemical modification of S. cerevisiae PEP carboxykinase with PLP-ADP and PLP-AMP. The results here described demonstrate the use of molecular modeling procedures when planning chemical modification of enzyme-active sites.
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  • 8
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    Molecular genetics and genomics 263 (2000), S. 81-89 
    ISSN: 1617-4623
    Keywords: Key words Flp recombinase ; Site-specific recombination ; Homologous recombination ; RAD52 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs). However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known. In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp. We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination. In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52. The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present. Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination.
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  • 9
    ISSN: 1617-4623
    Keywords: Key wordsYarrowia lipolytica ; Saccharomyces cerevisiae ; Ambient pH signalling ; Signal transduction ; Transmembrane protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In Yarrowia lipolytica, the transcription factor Rim101p mediates both pH regulation and control of mating and sporulation. Like its homologues PacC of Aspergillus nidulans and Rim101p of Saccharomyces cerevisiae, YlRim101p is activated by proteolytic C-terminal processing, which occurs in response to a signal transduced by a pathway involving several PAL gene products. We report here the cloning and sequencing of two of these genes, PAL2 and PAL3. PAL2 encodes a putative 632-residue protein with six possible transmembrane segments, which differs from the transmembrane proteins Rim9p of S. cerevisiae and PalI of A. nidulans, but is homologous to A. nidulans PalH and to the product of the ORF YNL294c, a predicted polypeptide of unknown function in S. cerevisiae. PAL3 encodes an 881-residue polypeptide that is homologous to PalF of A. nidulans and to a newly identified putative polypeptide of S. cerevisiae. Both PAL2 and PAL3 are expressed constitutively, regardless of ambient pH. Mutations in these genes affect growth at alkaline pH and sporulation in both Y. lipolytica and in S. cerevisiae. They affect invasiveness of haploid strains in S. cerevisiae only, and conjugation in Y. lipolytica only. These results highlight the conservation of the Pal pathway initially described in A. nidulans.
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  • 10
    ISSN: 1617-4623
    Keywords: Key wordsGAL regulon ; Transcription ; Saccharomyces cerevisiae ; Galactose suppression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A plasmid clone that suppresses galactose toxicity in a gal7 yeast strain has been isolated from a multicopy genomic DNA library. Molecular analysis revealed that the region responsible for the suppression of galactose toxicity corresponds to the ORF YPR030w, which was named MRG19. A CEN-based plasmid carrying the above ORF was unable to suppress the toxicity. Galactokinase activity was substantially reduced in cell extracts obtained from transformants bearing multiple copies of MRG19. Multiple copies of MRG19 were also able to suppress galactokinase expression driven by the CYC1 promoter but not the TEF1 promoter. Multiple copies of MRG19 could not suppress GAL1-driven galactokinase expression in a gal80 strain. However, MRG19-mediated suppression of CYC1-driven galactokinase expression was independent of GAL80 function. These results imply that multiple copies of MRG19 suppress galactokinase expression probably at the level of transcription. In agreement with this idea, multiple copies of MRG19 also suppress β-galactosidase expression driven by the GAL1 promoter in a GAL80-dependent manner. Disruption of MRG19 leads to an increase in the cell density at stationary phase in synthetic complete medium. MRG19 encodes a previously uncharacterised 124-kDa protein that shows no sequence homology to any known proteins.
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  • 11
    ISSN: 1617-4623
    Keywords: Key words Autonomously replicating sequence (ARS) ; Anti-bent DNA ; DNA structure ; Replication origin ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In order to better understand the involvement of the DNA molecule in the replication initiation process we have characterized the structure of the DNA at Autonomously Replicating Sequences (ARSs) in Saccharomyces cerevisiae. Using a new method for anti-bent DNA analysis, which allowed us to take into account the bending contribution of each successive base plate, we have investigated the higher-order structural organization of the DNA in the region which immediately surrounds the ARS consensus sequence (ACS). We have identified left- and right-handed anti-bent DNAs which flank this consensus sequence. The data show that this organization correlates with an active ACS. Analysis of the minimum nucleotide sequence providing ARS function to plasmids reveals an example where the critical nucleotides are restricted to the ACS and the right-handed anti-bent DNA domain, although most of the origins considered contained both left- and right-handed anti-bent DNAs. Moreover, mutational analysis shows that the right-handed form is necessary in order to sustain a specific DNA conformation which is correlated with the level of plasmid maintenance. A model for the role of these individual structural components of the yeast replication origin is presented. We discuss the possible role of the right-handed anti-bent DNA domain, in conjunction with the ACS, in the process of replication initiation, and potentialities offered by the combination of left- and right-handed structural components in origin function.
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  • 12
    Electronic Resource
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    Molecular genetics and genomics 263 (2000), S. 877-888 
    ISSN: 1617-4623
    Keywords: Key words Staurosporine ; Vacuolar-type proton pumping ATPase ; Vacuolar protein sorting ; ATP-binding cassette transporter ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mutations at several loci affect the sensitivity of the yeast Saccharomyces cerevisiae to staurosporine. We report here the characterization of novel staurosporine- and temperature-sensitive mutants (stt). Cloning and integration mapping showed that the genes STT2/STT6, STT5, STT7, STT8 and STT9 are allelic to VPS18, ERG10, GPI1, VPS34 and VPS11, respectively. The products of ERG10 and GPI1, respectively, catalyze mevalonate and glycosyl phosphatidylinositol anchor synthesis, while VPS18 and VPS11 genes belong to the class C VPS (Vacuolar Protein Sorting) genes, and the VPS34 gene is classified as a class D VPS. Therefore, staurosporine sensitivity is affected by ergosterol and glycolipid biosynthesis and by vacuolar functions. We found that other vps mutants belonging to classes C and D exhibit staurosporine sensitivity, and that they show calcium sensitivity and fail to grow on glycerol as the sole carbon source; both of the last two characteristics are shared by vacuolar H+-ATPase mutants (vma). As vma mutants were also found to show staurosporine-sensitive growth, staurosporine sensitivity is likely to be affected by acidification of the vacuole. Moreover, wild type yeast cells are more sensitive to staurosporine in alkaline media than in acidic media, suggesting that staurosporine is exported from the cytosol by H+/drug antiporters. Pleiotropic drug resistance (PDR) genes also provide some resistance to staurosporine, because Δpdr5, Δsnq2 and Δyor1 strains are more sensitive to staurosporine than the wild-type strain. This suggests that staurosporine is also exported by the ATP-binding cassette (ABC) transporters on the plasma membrane. vma mutants and vps mutants of classes C and D vps are sensitive to hygromycin B and vanadate, while ABC transporter-depleted mutants do not show such sensitivity, indicating that two systems differ in their ability to protect the cell against different types of drug.
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  • 13
    ISSN: 1617-4623
    Keywords: Key words DNA repair ; Helix-hairpin-Helix motif ; Methylmethane sulfonate (MMS) ; Saccharomyces cerevisiae ; UV radiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The gene MUS81 (Methyl methansulfonate, UV sensitive) was identified as clone 81 in a two-hybrid screen using the Saccharomyces cerevisiae Rad54 protein as a bait. It encodes a novel protein with a predicted molecular mass of 72,316 (632 amino acids) and contains two helix-hairpin-helix motifs, which are found in many proteins involved in DNA metabolism in bacteria, yeast, and mammals. Mus81p also shares homology with motifs found in the XPF endonuclease superfamily. Deletion of MUS81 caused a recessive methyl methansulfonate- and UV-sensitive phenotype. However, mus81Δ cells were not significantly more sensitive than wild-type to γ-radiation or double-strand breaks induced by HO endonuclease. Double mutant analysis suggests that Rad54p and Mus81p act in one pathway for the repair of, or tolerance to, UV-induced DNA damage. A complex containing Mus81p and Rad54p was identified in immunoprecipitation experiments. Deletion of MUS81 virtually eliminated sporulation in one strain background and reduced sporulation and spore viability in another. Potential homologs of Mus81p have been identified in Schizosaccharomyces pombe, Caenorhabditis elegans and Arabidopsis thaliana. We hypothesize that Mus81p plays a role in the recognition and/or processing of certain types of DNA damage (caused by UV and MMS) during repair or tolerance processes involving the recombinational repair pathway.
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  • 14
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    Journal of bioenergetics and biomembranes 32 (2000), S. 391-400 
    ISSN: 1573-6881
    Keywords: ATP synthase ; F1-ATPase ; Saccharomyces cerevisiae ; petite mutants ; epistasis ; mitochondrion ; pet mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The mitochondrial ATP synthase is a molecular motor that drives the phosphorylation ofADP to ATP. The yeast mitochondrial ATP synthase is composed of at least 19 differentpeptides, which comprise the F1 catalytic domain, the F0 proton pore, and two stalks, oneof which is thought to act as a stator to link and hold F1 to F0, and the other as a rotor.Genetic studies using yeast Saccharomyces cerevisiae have suggested the hypothesis thatthe yeast mitochondrial ATP synthase can be assembled in the absence of 1, and even 2, ofthe polypeptides that are thought to comprise the rotor. However, the enzyme complexassembled in the absence of the rotor is thought to be uncoupled, allowing protons to freelyflow through F0 into the mitochondrial matrix. Left uncontrolled, this is a lethal process andthe cell must eliminate this leak if it is to survive. In yeast, the cell is thought to lose ordelete its mitochondrial DNA (the petite mutation) thereby eliminating the genes encodingessential components of F0. Recent biochemical studies in yeast, and prior studies in E. coli,have provided support for the assembly of a partial ATP synthase in which the ATP synthaseis no longer coupled to proton translocation.
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  • 15
    ISSN: 1572-9699
    Keywords: electron microscopy ; killer effect ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A mesophilic wine yeast, Saccharomyces cerevisiae CSIR Y217 K − R − was subjected to the K2 killer effect of Saccharomyces cerevisiae T206 K + R + in a liquid grape medium. The lethal effect of the K2 mycoviral toxin was confirmed by methylene blue staining. Scanning electron microscopy of cells from challenge experiments revealed rippled cell surfaces, accompanied by cracks and pores, while those unaffected by the toxin, as in the control experiments, showed a smooth surface. Transmission electron microscopy revealed that the toxin damaged the cell wall structure and perturbed cytoplasmic membranes to a limited extent.
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  • 16
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    Antonie van Leeuwenhoek 78 (2000), S. 187-194 
    ISSN: 1572-9699
    Keywords: cAMP ; pseudohyphae ; Saccharomyces cerevisiae ; stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In Saccharomyces cerevisiae pseudohyphae formation may be triggered by nitrogen deprivation and is stimulated by cAMP. It was observed that even in a medium with an adequate nitrogen supply, cAMP can induce pseudohyphal growth when S. cerevisiae uses ethanol as carbon source. This led us to investigate the effects of the carbon source and of a variety of stresses on yeast morphology. Pseudohyphae formation and invasive growth were observed in a rich medium (YP) with poor carbon sources such as lactate or ethanol. External cAMP was required for the morphogenetic transition in one genetic background, but was dispensable in strain Σ1278b which has been shown to have an overactive Ras2/cAMP pathway. Pseudohyphal growth and invasiveness also took place in YPD plates when the yeast was subjected to different stresses: a mild heat-stress (37 °C), an osmotic stress (1 m NACl), or addition of compounds which affect the lipid bilayer organization of the cell membrane (aliphatic alcohols at 2%) or alter the glucan structure of the cell wall (Congo red). We conclude that pseudohyphal growth is a physiological response not only to starvation but also to a stressful environment; it appears to require the coordinate action of a MAP kinase cascade and a cAMP-dependent pathway.
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  • 17
    ISSN: 1573-5028
    Keywords: gene expression ; heterologous expression ; H+/hexose symporter ; Lycopersicon esculentum ; quantitative PCR ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H+/hexose symporter. The K m of the symporter for glucose is 45 μM.
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  • 18
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    Chemistry of natural compounds 36 (2000), S. 88-89 
    ISSN: 1573-8388
    Keywords: Saccharomyces cerevisiae ; yeast invertase ; active enzyme
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The substrate specificity of purified yeast invertase isolated fromSaccharomyces cerevisiae in transglycosylation reactions was determined. The enzyme is specific for primary alcohols. The yeast activity is a function of the alkyl length and substrate hydrophobicity (n-butyl, isobutyl, isoamyl alcohols).
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  • 19
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    Cellular and molecular life sciences 40 (1984), S. 1159-1161 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; 5-trifluoromethyl-6-àzauracil ; yeast cell cultures ; cell division ; inhibition of
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Cell division, as studied in asynchronous cultures of yeast cells, is sensitive to 5-trifluoromethyl-6-azauracil (F3CAzU). Under defined conditions (10 mmoles l−1 F3CAzU) this compound blocks immediately and completely the process of cell division. Using synchronized cells, the time-point at which division process of yeast cell can be inhibited by F3CAzU has been determined. The inhibitor effect of this compound is completely reversed by thymine, thymidine and uracil.
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  • 20
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    Current genetics 8 (1984), S. 559-566 
    ISSN: 1432-0983
    Keywords: DNA repair ; Saccharomyces cerevisiae ; Cloning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Three overlapping plasmids were isolated from a YEp24 library, which restore Rad+ functions to rad6-1 and rad6-3 mutants. Different subclones were made and shown to integrate by homologous recombination at the RAD6 site on chromosome VII, thus verifying the cloned DNA segments to be the RAD6 gene and not a suppressor. The gene resides in a 1.15 kb fragment, which restores Rad+ levels of resistance to U.V., MMS and γ-rays to both rad6-1 and rad6-3 strains. It also restores sporulation ability to rad6-1 diploids. Integrative deletion of the RAD6 gene was shown not to be completely lethal to the yeast. Our results suggest that the RAD6 gene has some cell cycle-specific function(s), probably during late S phase.
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  • 21
    ISSN: 1432-0983
    Keywords: α-Pheromone-inactivating glycoproteins ; bar1-1 ; Barrier proteins ; Purification ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two kinds of a-mating-type-specific proteins inactivating α pheromone (α factor) were purified from heat shock extract of MATa cells. Their molecular weights were estimated to be 400,000 and 200,000 by gel filtration. Both proteins were detected in MATa SST1 cells but not in MATα SST1, MATa sst1-1 and MATa/MATα SST1/SST1 cells. In addition, the proteins were detected in matα2-1 SST1 cells but not in matα1-2 SST1 cells. From these results, it is concluded that these proteins are synthesized under the control of the SST1 gene and responsible for the Barrier action of MATa cells. The relationship of these proteins to the secreted Barrier protein having a higher molecular weight is discussed.
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  • 22
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; TRP3 gene ; Deletion analysis ; Enzyme function
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two sets of deletions, entering the TRP3 gene of Saccharomyces cerevisiae from the 3′- and the 5′-end were constructed. Complementation analysis with chromosomal trp3A, trp3B and trp3C mutations was done by introducing the 3′- and 5′-truncated gene on a multicopy 2 μm-vector. The N-terminal glutamine amido transferase function is encoded by a DNA fragment of 600–700 bp, and the C-terminal indole-3-glycerol-phosphate synthase function by a DNA fragment of about 900 bp, whereas both functions together are encoded by a contiguous DNA fragment of about 1,500 bp. The bi functional TRP3-peptide thus could be dissected into two catalytically independent peptides in vivo. For the indole-3-glycerol-phosphate synthase activity, independent catalytic activity was also demonstrated in vitro: deletions entering the TRP3 gene from the 5′-end, and lacking large parts of the sequence coding for the glutamine amidotransferase function, still are able to ex press a peptide exhibiting functional indole-3-glycerol phosphate synthase activity in vitro. Deletion plasmids pME505·De1C102·2μm and DelC10·2μm exhibited shorter TRP3 transcripts according to the deleted DNA-fragments (150 and 426 by respectively) but yielded peptides of invariable Mr of 35,000 d. Transcription and translation of these peptides, which probably represent the independently folding indole-3-glycerol-phosphate synthase core are discussed.
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  • 23
    ISSN: 1432-0983
    Keywords: Cephalosporium acremonium ; Mitochondrial DNA ; Autonomous replication sequence ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A fragment of DNA which functions as an autonomous replication sequence in yeast was cloned from Cephalosporium acremonium. Mitochondrial DNA (mtDNA) was isolated from an industrial strain of C. acremonium (08G-250-21) highly developed for the production of the antibiotic, cephalosporin C. Size, 27 kb, and restriction pattern indicated this DNA was identical to mtDNA previously isolated (Minuth et al. 1982) from an ancestral strain (ATTC 14553) which produces very low amounts of cephalosporin C. A 1.9 kb Pst1 fragment of the Cephalosporium mtDNA was inserted into a Pst1 site of the yeast integrative plasmid, Ylp5, to produce a 7.5 kb plasmid, designated pPS1. The structure of pPS1 was verified by restriction analysis and hybridization. PS1 transformed Saccharomyces cerevisiae (DBY-746) to uracil prototrophy at a frequency of 272 transformants/μg DNA. Transformation frequencies of 715 transformants/μg DNA and zero were obtained for the replicative plasmid, YRp7, and the integrative plasmid YIp5, respectively. Southern hybridization and transformation of E. coli by DNA from yeast transformed by pPS1 verified that pPS1 replicates autonomously in yeast. The uracil-independent pPS1-yeast transformants were mitotically unstable. The average retention of pPS1 after three days growth in selective and non-selective medium was 4.5% and 0.4%, respectively, compared to retentions of 4.6% and 0.5% for YRp7. The properties of pPS1 were compared to those of a related plasmid, pCP2. pCP2 was constructed (Tudzynski et al. 1982) by inserting the C. acremonium 1.9 kb Pst1 fragment into the yeast integrative plasmid, pDAM1.
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  • 24
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; 5-aminolevulinate synthase ; Cloned gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1–5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1–5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.
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  • 25
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    Current genetics 9 (1984), S. 107-111 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; 2 μm minichromosomes ; Metrizamide gradients
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    Topics: Biology
    Notes: Summary Two micrometer minichromosomes from Saccharomyces cerevisiae were isolated without detergent using metrizamide gradients. 2 μm minichromosomes showed a lower density in metrizamide gradients relative to genomic chromatin. Our results suggest a lower ratio of proteins to DNA in 2-μm minichromosomes as compared with genomic chromatin. The procedure described herein yields minichromosomes free of cellular chromatin and ribosomal protein contamination. This method may be useful for the isolation and characterization of other yeast minichromosomes.
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  • 26
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    Current genetics 8 (1984), S. 81-84 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Mitochondrial genes ; Vegetative segregation ; Uniparental inheritance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Zygotes of Saccharomyces cerevisiae that are heteroplasmic for mitochondrial alleles produce diploid progeny that are homoplasmic for one allele or the other, judged by the criterion that upon further subcloning they produce daughter cells of only one phenotype or the other. Here we show that when such cells are subjected to strong selection for the missing allele, it cannot be detected, so that it is probably not present in even a single copy.
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  • 27
    ISSN: 1432-0983
    Keywords: Iso-1-cytochrome c ; Saccharomyces cerevisiae ; Heme ; Transcription
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    Topics: Biology
    Notes: Summary A Saccharomyces cerevisiae mutant (hem1 cycl-1) was transformed with plasmids bearing a chromosomal centromer (CEN3) and a 2 μm DNA replication origin. In one of the plasmids a functional CYC1 gene was present, in a second plasmid an XhoI fragment located between bases -245 and -678 upstream from the translation initiation codon had been deleted, in a third plasmid this region had been inverted. Results of hybridization experiments carried out with mRNA isolated from heme-deficient and heme-containing transformants indicated that heme controls transcription of the CYC1 gene and that DNA sequences located within the upstream XhoI fragment are involved in activation of the gene by heme.
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  • 28
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; TRP3 gene ; Sequence analysis ; Enzyme function
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    Topics: Biology
    Notes: Summary The structure and function of the TRP3 gene of Saccharomyces cerevisiae were analyzed. Subcloning of an original 4.8 kb BamHI DNA fragment, carrying the yeast TRP3 gene, allowed for a localization of the gene on a 2.5 kb ClaI/BamHI fragment. Transcription was found to proceed from the ClaI site towards the BamHI site. Three major transcription start sites were determined at positions −92, −87, and −81 by S1-mapping. The synthesis of the TRP3 gene is regulated by the general control, and was found to take place- at the transcriptional level. The sequence of the 5′-noncoding region up to position −400 and part of the coding region to position 840 were determined. The 5′-noncoding region contains sequences common to most amino acid biosynthetic genes known so far, namely a presumptive ribosome binding site, “Goldberg-Hogness boxes”, and a consensus sequence, possibly involved in the general control. For the coding region a single open reading frame was found. The deduced amino acid sequence was aligned with homologous amino acid sequences of Neurospora crassa, Pseudomonas putida and Escherichia coli. The exceptionally high homology (40–60%) between these sequences led us to postulate that the TRP3 gene product is of the structure NH2-glutamine amidotransferase-indole-3-glycerol-phosphate synthase-COOH.
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  • 29
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Cloning ; Suppressor
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    Notes: Summary A plasmid, pYsup1-1, containing a DNA fragment able to suppress the recessive mutant phenotype of the suppressor locus sup1 (allele sup1-ts36) of Saccharomyces cerevisiae was isolated from a bank of yeast chromosomal DNA cloned in cosmid p3030. The complementing gene was localized on a 2.6 kb DNA fragment by further subcloning. Evidence is presented that the cloned DNA segment codes for the sup1 structural gene (chromosome IIR).
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  • 30
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    Current genetics 8 (1984), S. 575-580 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Candida utilis ; Protoplast fusion
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    Topics: Biology
    Notes: Summary Auxotrophic mutants of Saccharomyces cerevisiae and Candida utilis were hybridized through protoplast fusion. Spontaneous, UV- and FPA-induced mitotic segregation indicated that after cell fusion, exclusion of the S. cerevisiae nucleus or nuclear fusion followed by preferential loss of S. cerevisiae chromosomes can take place. Some of the hybrids were stable. One of them, expressed mating and sporulation functions of the S. cerevisiae parent. Thus, markers from both parents could be recovered as mitotic and meiotic segregants.
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  • 31
    ISSN: 1432-072X
    Keywords: Agglutination substance ; α Pheromone ; Cell cycle ; Ethyl N-phenylcarbamate ; Mating reaction ; Microtubules ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract Effects of ethyl N-phenylcarbamate (EPC) on the mating reaction of Saccharomyces cerevisiae were studied, with special attention on the effect on the α pheromone action. EPC inhibited zygote formation at a concentration which promoted induction of sexual agglutinability. EPC enhanced agglutinability induction by α pheromone, but inhibited α-pheromone-induced formation of large pearshaped cells in a mating type. The enhancement of agglutinability induction was accompanied with increased production of a agglutination substance and inhibition of α pheromone inactivation. EPC arrested the cell cycle of a cells probably in the step controlled by CDC19, CDC35, cAMP etc., just before the step controlled by CDC28, α pheromone etc.
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  • 32
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Glucan synthetase ; EDTA ; Magnesium ; Sucrose ; Fluoride
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    Notes: Abstract Yeast β(1–3) glucan synthetase is stimulated and stabilized by EDTA. Sucrose protects the enzyme from selfinactivaton. Preincubation of cell free extracts at low sucrose concentrations indicates a slow transition of the enzyme towards dissociation. Transition kinetics at 30° C and 0° C in the presence and in the absence of sucrose are interpreted assuming that a subunit is thermolabile in the free state and that sucrose increases its stability. Magnesium is deletereous for glucan synthetase in cell-free extracts. Chaotropic agents inactivate glucan synthetase according to their capacity to solubilize and depolymerize biological compounds. Fluoride plays a special role in the activation of glucan synthetase. Its action appears to be dependent on the presence of GTP (or other nucleotides). The role of all these agents on the activity and stability of the enzyme is interpreted in a unified scheme.
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  • 33
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    Archives of microbiology 137 (1984), S. 357-361 
    ISSN: 1432-072X
    Keywords: Yeast ; Saccharomyces cerevisiae ; Killer toxin ; Extracellular glycoprotein
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    Topics: Biology
    Notes: Abstract A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of Mr=16,000. The amino acid composition of the toxin type K2 of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.
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  • 34
    ISSN: 1432-072X
    Keywords: Agglutination substance ; Cell-cell recognition ; Glycoprotein ; Mating ; Saccharomyces cerevisiae ; Sexual agglutinability ; Yeast
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    Topics: Biology
    Notes: Abstract An a-mating-type-specific substance responsible for sexual agglutination was purified to 397-times in specific activity (units/mg protein) from the cytoplasm of a-mating type cells. The purified substance gave a single band stained with PAS reagent but not with both Coomassie brilliant blue and silver staining reagent by polyacrylamide gel electrophoresis in the presence of 8 M urea. However, incorporation of [35S]methionine and Lowry reaction clearly indicate that the substance is a glycoprotein. The substance specifically masked sexual agglutinability of cells of the opposite mating type α, indicating univalent action. The substance is a glycoprotein with a carbohydrate content of 90%, a pI of 4.5, and a molecular weight of 130,000. The substance was inactivated by 2-mercaptoethanol and proteolytic enzymes but not by glycolytic enzymes. The substance formed a complementary complex having no biological activity when mixed with α-agglutination substance from the wall or cytoplasm of α-cells in vitro.
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  • 35
    ISSN: 1432-072X
    Keywords: Glycoprotein ; Inducible strains ; Saccharomyces cerevisiae ; Sexual agglutinability ; Tunicamycin
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    Notes: Abstract Effects of tunicamycin (TM) on the sexual agglutinability and zygote formation of Saccharomyces cerevisiae were studied using the two kinds of haploid strains, inducible and constitutive for sexual agglutinability. Induction of sexual agglutinability by opposite mating type sex pheromone of inducible strains was inhibited by TM in α mating type but not in a mating type. The recovery by temperature-shift-down from the temperature-suppressed sexual agglutinability of constitutive strains was enhanced by TM in a mating type but rather inhibited in α mating type. Pretreatment with TM of constitutive strains enhanced sexual agglutinability in a mating type but not in α mating type. The above-mentioned a-mating-type-specific agglutinability-enhancing actions of TM were discussed in relation to the action mechanism of α pheromone which induces or enhances the sexual agglutinability of a cells. Zygote formation was inhibited by TM in both constitutive and inducible strains at concentrations which showed only partially inhibitory effect on sexual agglutinability.
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  • 36
    ISSN: 1573-1561
    Keywords: Leptopilinaheterotoma ; Hymenoptera ; Eucoilidae ; Saccharomyces cerevisiae ; host-habitat searching ; chemoreception ; fermentation products ; ethanol ; ethyl acetate ; acetaldehyde
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Chemical stimuli play an important role in the process of searching for a host habitat by parasitic wasps. Volatile compounds originating from host habitats and/or hosts are the cues that enable such a location.Leptopilina heterotoma, a larval parasite ofDrosophila, is attracted to the food of its host, baker's yeast. Analysis of the fermentation products of baker's yeast, using a mass spectrometer, and olfactometer studies indicate that three fermentation products of this yeast, the main component of the host habitat in our laboratory, attractL. heterotoma: ethanol (5%), ethyl acetate (10−2, 10−3%), and acetaldehyde (1%). A combination of these three compounds, however, cannot compete with baker's yeast in attracting the parasites. Thus other factors, such as different compounds, concentrations, and/or combinations, also, play a role and remain to be tested.Leptopilina heterotoma does not use host-related olfactory cues in long-distance habitat location as it cannot distinguish between host habitat and host habitat with hosts.
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  • 37
    ISSN: 1573-6881
    Keywords: H+-ATPase complex ; assembly defect ; Saccharomyces cerevisiae ; mitochondrial biogenesis ; membrane association
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    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H+-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H+-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized β-subunit of the H+-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H+-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.
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  • 38
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    European journal of nutrition 22 (1983), S. 205-212 
    ISSN: 1436-6215
    Keywords: Schwermetallwirkung ; Malatdehydrogenase ; Glutamatdehydrogenase ; Glycerinaldehyd-3-phosphatdehydrogenase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Medicine
    Description / Table of Contents: Summary The difference between cadmium, zinc, lead, and mercury in regard of their effects on the activity of the enzymes tested is very slight. Concentrations higher than 10−5 M reduce significantly the activity of the enzymes, and concentrations of approximately 10−3 M inhibit it completely. An increase of the activity cannot be detected. The addition of combinations of cadmium, zinc, and lead results in a summing up of the toxic effects, whereas the interaction between mercury and the other three heavy metals shows a cumulative effect, which is appointed nearly completely by the heavy metal more toxic. The findings suggest that under in-vitro conditions there exists a direct interaction between the heavy metals and the enzymes.
    Notes: Zusammenfassung Die vier Schwermetalle Cadmium, Zink, Blei und Quecksilber unterscheiden sich in ihrer Wirkung auf die Aktivität der untersuchten Enzyme nur sehr wenig. Konzentrationen über 10−5 M vermindern die Enzymaktivität signifikant, und Konzentrationen von etwa 10−3 M unterbinden sie völlig. Eine Steigerung der Enzymaktivität läßt sich nicht feststellen. Die Zugabe von Cadmium-, Zink- und Bleikombinationen führt zu einer Addition der toxischen Effekte, während bei der Interaktion zwischen Quecksilber und den anderen drei Schwermetallen die Gesamtwirkung fast ausschließlich durch das stärker hemmende Schwermetall allein bestimmt wird. Die erhaltenen Ergebnisse lassen vermuten, daß es unter Invitro-Bedingungen zu einer direkten Wechselwirkung zwischen den Schwermetallen und den Enzymen kommt.
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  • 39
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    Current genetics 7 (1983), S. 165-166 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Cephalosporium acremonium ; Mitochondrial hybrid vector ; Nuclear association
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    Topics: Biology
    Notes: Summary The hybrid vector pCP2, consisting of the bacterial plasmid pBR325, the nuclear gene Leu-2 of Saccharomyces cerevisiae and a fragment of mitochondrial DNA from Cephalosporium acremonium, was found to associate with the nucleus in a transformed strain of Saccharomyces cerevisiae. This was inducted by (1) efficient expression of the Leu-2 gene as evidenced by a short generation time on selective medium; (2) independence of Leu-2 gene expression from mitochondrial protein synthesis, since pCP2 was shown to replicate and to be expressed in petite mutants; (3) association of pCP2 with isolated DNA from nuclei as proved by transformation experiments with E. coli.
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  • 40
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; G1 cdc mutants ; tα-factor
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    Topics: Biology
    Notes: Summary Mutants in four G1 cdc strains of Saccharomyces cerevisiae were isolated which failed to show division arrest in the presence of α-factor. The cell cycle properties, terminal arrest morphology and mating competence of these mutants at the restrictive temperature were examined. The G1 specific arrest of the cdc 36 and cdc39 mutants is dependent upon the availability of an intact mating factor response system in Mat a cells. Cdc28 and cdc37 mutants exert their cell cycle blocks independently of the mating factor pathway. It is likely that the nature of the primary growth defect in cdc36 and cdc39 mutants is such that the α-factor pathway is activated in the absence of the pheromone at the restrictive temperature and that G1 arrest is a secondary consequence of a non-cycle specific event in such mutants.
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  • 41
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    Current genetics 7 (1983), S. 235-237 
    ISSN: 1432-0983
    Keywords: DNA replication ; Shuttle vectors ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Summary Mitotic segregation of three 2 μm-pBR322 chimaeric plasmids (YEp6, YEp21, and YEp24) was studied in yeast. Each displayed a characteristic rate of loss: YEp6 was lost at approximately twice the rate of YEp21 and YEp24. The loss rates were not significantly increased when two chimaeric plasmids were coresident, nor was the endogenous 2 μm plasmid itself displaced. Therefore these plasmids appear to be compatible in yeast.
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  • 42
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; DEL1 ; rad ; ste7
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In DEL1 strains of the yeast, Saccharomyces cerevisiae, the iso-1-cytochrome c (CYC1) region is flanked on either side by Tyl elements in direct orientation which promote cyc1 deletions of the bracketed DNA in the haploid cell. In this study, we asked which genes might control this event by testing the possibility that the DEL1 mutation mechanism requires an enzyme (or enzymes) that is also utilized in the repair of damaged DNA. To this end, we independently coupled eight repair mutations, rad3–2, rad4–4, rad6–1, rad6–3, rad9–1, rev3–1, rad50–1, and rad51-1, toDEL1 and asked whether DEL1 was still functional. We found that none of these rad mutations significantly affects the mutation frequency of 10−6-10−5 established in DEL1 strains for the CYC1 locus. Furthermore, we determined that ste7, a temperature-sensitive sterile allele known to alter gene regulation in Ty-mediated mutations, is not required for DEL1 function. Finally, DEL1 is not temperature-sensitive at 23° or 37 °C.
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  • 43
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    Current genetics 7 (1983), S. 369-377 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Isoenzymes ; Induction
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    Topics: Biology
    Notes: Summary Tetrad analysis indicates that α-isopropylmalate synthase activity of yeast is determined by two separate genes, designated LEU4 and LEU5. LEU4 is identified as a structural gene. LEU5 either encodes another α-isopropylmalate synthase activity by itself or provides some function needed for the expression of a second structural gene. The properties of mutants affecting the biosynthesis of leucine and its regulation suggest that the expression of LEU1 and LEU2 (structural genes encoding isopropylmalate isomerase and β-isopropylmalate dehydrogenase, respectively) is controlled by a complex of a-isopropylmalate and a regulatory element (the LEU3 gene product). Similarities and differences between yeast and Neurospora crassa with respect to leucine biosynthesis are discussed.
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  • 44
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    Current genetics 7 (1983), S. 393-397 
    ISSN: 1432-0983
    Keywords: Trehalose ; Glycogen ; Sporulation ; Germination ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Summary Mutants with specific lesions were used to differentiate between the functions of glycogen and trehalose in S. cerevisiae. Diploids which harbor the glc1/glc1 mutation depend upon the phosphorylated, less active form of glycogen synthase and show a more active, phosphorylated form, of the enzyme trehalase. These conditions are due to a lesion in the regulating subunit of the cAMP-dependent protein kinase. Such cells are unable to sporulate. Diploids which contain the sst1/sst1 mutation have normal glycogen metabolism but their trehalose-6-phosphate synthase is not active. Such strains sporulate but germination is poor and only one-spore tetrads are formed. These results confirm that glycogen is needed to trigger sporulation while trehalose plays a role in the germination process. Different systems, I and II, of trehalose accumulation were proposed. System I would require the UDPG-linked trehalose synthase, whereas system II would constitute an alternative pathway, specifically induced or activated by the expression of a MAL gene. The presence of system II in its constitutive form in the constructed diploids would favour trehalose synthesis during growth on glucose, however, it did not overcome the glycogen deficiency during sporulation nor the lack of trehalose for germination. It seems that only system I, namely trehalose 6-P-synthase, plays a role in the germination process.
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  • 45
    ISSN: 1432-0983
    Keywords: Oversecretion mutants ; Protease defect ; Wall glucan defect ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two chromosomal mutations in yeast that result in oversecretion of the K1 killer toxin protein were examined. A recessive mutation in gene ski5 appears to lead to toxin oversecretion through a defect in a cell surface, PMSF-inhibited protease. A wild type killer strain degraded toxin following synthesis, and degradation could be partially prevented by addition of PMSF to the growth medium. The ski5 mutation caused an approximate ten fold oversecretion of toxin, similar to that seen in a PMSF-treated wild type culture, and no increased oversecretion in the presence of PMSF. The ski5 mutation caused oversecretion of other low molecular weight secreted proteins and appeared to oversecrete the α-factor pheromone, as judged by activity tests. The ski5 mutation was complemented by mutations in ski genes 1–4, and the mutant was not supersensitive to mating pheromones or K2 killer toxin. We also examined killer strains with a mutation in the nuclear gene krel which results in a defective (1→6)-β-D-glucan cell wall receptor for killer toxin. Such strains oversecrete toxin into the growth medium, but also, unexpectedly, oversecrete most other secreted proteins. The defect in (1→6)-β-D-glucan in these mutants appears to perturb the partitioning of secreted proteins between the cell wall and the medium.
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  • 46
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    Current genetics 7 (1983), S. 427-431 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; DNA ; Alkaline elution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The method of analysis of DNA in mammalian cells by alkaline elution from filters (Kohn et al. 1974) was adapted for studies on yeast DNA. By this technique spheroplasts obtained from yeast cells are lysed on filters and single-stranded DNA fragments selectively eluted by alkaline solutions. The procedure was applied to monitor the occurrence of replication intermediates and production of DNA single-strand breakage by MMS, and its repair in growth medium.
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  • 47
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    Current genetics 7 (1983), S. 433-438 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Yeast transformation ; Yeast autonomously replicating sequences ; Ribosomal RNA genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have previously demonstrated that the loss of Rcp-CEN3, a centromeric plasmid containing yeast rDNA autonomously replicating sequences (ARS) is as high as around 50% per generation for most yeast strains. In this study we have attempted to elucidate mechanisms underlying the high mitotic instability of Rcp-CEN3. For this purpose a tandem duplication of a rDNA ARS was constructed in Rcp-CEN3. The new plasmid having two ARSs possesses a markedly higher mitotic stability as compared to a monoARS Rcp-CEN3. The mitotic stability of this centromere-containing plasmid which has two replicators corresponds to the calculated value for the mitotic stability of two monoARS plasmids Rcp-CEN3 in given cells. Genetic analysis has demonstrated that both plasmids having one or two ARSs are maintained in the single copy state. These results demonstrate that the mitotic instability of centromeric plasmid Rcp-CEN3 carrying a rDNA ARS is associated with the absence of stringent control of replication from the rDNA ARS. A possible mechanism of replication of the chromosomal rDNA repeats in yeast is discussed in the light of this data.
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  • 48
    ISSN: 1432-072X
    Keywords: α Pheromone ; Cell cycle ; G1 arrest ; Hansenula wingei ; Saccharomyces cerevisiae ; Shmoo
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The cell cycle of a (5) mating type cells of Hansenula wingei was arrested in the G1 phase by α pheromone of Saccharomyces cerevisiae but not by α(21) pheromone of H. wingei, although both the α pheromones are known to induce sexual agglutination ability of a mating type cells of H. wingei. Cells of α mating type of H. wingei became shmooed or arrested in the G1 phase in response to neither a pheromone of H. wingei nor α pheromone of S. cerevisiae.
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  • 49
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    Archives of microbiology 134 (1983), S. 64-67 
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Anaerobic growth ; Hungate technique ; Tween 80 ; Ergosterol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Saccharomyces cerevisiae, Nontrachet strain 522 was successfully grown anaerobically on various glucose concentrations in Yeast Nitrogen Base (YNB) medium (pH 3.5) prepared under an atmosphere of carbon dioxide (CO2). This growth occurred in the absence of Tween 80 and ergosterol. The medium, prepared using the Hungate technique for cultivation of strictly anaerobic bacteria, contained the reducing agent cysteine·HCl·H2O (0.03%). Anaerobic growth was stimulated by the addition of Tween 80 and ergosterol to the anaerobic medium.
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  • 50
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    Archives of microbiology 134 (1983), S. 171-174 
    ISSN: 1432-072X
    Keywords: Acetate growth medium ; Anti-microtubule agent ; Bud initiation ; Ethyl N-phenylcarbamate ; Meiosis ; Mitotic cell cycle ; Saccharomyces cerevisiae ; Sporulation induction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract When diploid cells of Saccharomyces cerevisiae were incubated in acetate growth media containing 2.5 mM ethyl N-phenylcarbamate (EPC), bud initiation was inhibited preferentially, and eventually overgrown, unbudded cells accumulated. During subsequent incubation, meiosis and ascospore formation occurred at high frequencies. The behavior of EPC-treated cells was essentially the same as that of cells transferred to a starvation sporulation medium. EPC thus has a pronounced effect on the mitotic growth of yeast cells, which leads to meiotic development. Our observations indicate that EPC has a decisive function in the initiation of meiosis in rich growth media.
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  • 51
    ISSN: 1432-072X
    Keywords: Nucleotide pools ; Continuous cultivation ; Synchronized growth ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract High pressure liquidd chromatography has been used to study the acid soluble nucleotide pool of Saccharomyces cerevisiae under different conditions of growth. ATP, ADP, AMP, NAD, GTP, UTP, UDP, CTP, CDP, and UDP-sugars plus UMP could be separated and were found in concentrations higher than 0.1 μmol per g yeast cell dry weight (=detection limit). During glucose-limited continuous culture the levels of individual nucleotides depended on the growth rate, which was most pronounced with pyrimidine (uridine, cytidine) nucleotides. The energy charge (E.C.) remained high (0.9) at all growth rates (0.07–0.3 h-1). During synchronized growth at a constant growth rate (0.11 h-1) almost all nucleotide levels and the E.C. remained at constant values with the only exception of UDP-sugars and UMP of which increased levels were found during the phase of budding. Under conditions of metabolic stress (addition of antimycin A, deoxyglucose plus iodoacetate) pronounced changes in the levels of purine (adenine and guanine) nucleotides and the E.C. were observed. All other nucleotides were less influenced by these conditions. Only under these conditions IMP accumulation was observed. The results strongly argue against the significance of purine nucleotide or E.C. measurements under viable conditions. In contrast, changes in the levels of pyrimidine nucleotides seem to be indicative of changes in the flux through the metabolic pathways where they act as coenzymes.
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  • 52
    ISSN: 1432-1424
    Keywords: electron probe X-ray microanalysis ; Saccharomyces cerevisiae ; ethidium ; brontophenol blue ; cationic dye ; cytolysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary K+ efflux provoked by ethidium proceeds partially as an all-or-none effect by which the diffusion barrier for K+ is disrupted and partially from still intact cells, presumably by exchange against ethidium. This is shown by the application of an electron probe microanalysis X-ray technique by which the K+ content of a number of individual cells is analyzed.
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  • 53
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    Applied microbiology and biotechnology 16 (1982), S. 75-80 
    ISSN: 1432-0614
    Keywords: Immobilization of yeast ; Saccharomyces cerevisiae ; Ethanol production
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.
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  • 54
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Multiple mutants of DNA repair ; Sensitivity to nitrogen mustard and to radiation ; Thermoconditional DNA repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Three haploid yeast mutants (snm) sensitive or thermoconditionally sensitive to the DNA cross-linking agent nitrogen mustard (HN2) were crossed with four rad strains representing mutations in the three pathways of DNA dark repair. The resulting haploid double and triple mutant strains were tested for their sensitivity to UV, HN2 and HN1. From the observed epistatic or synergistic interactions of the combinations of mutant alleles we could derive the relation of the SNM1 and SNM2 genes to the postulated repair pathways. Alleles snm1-1 and snml-2 ts were found epistatic to genes of the rad3 group, whereas snm2-1 ts was epistatic to rad6. The snm1 and snm2 mutant alleles interacted synergistically. From these data it is concluded that the SNM1 gene product plays a cross-link specific role in excision repair while the SNM2 gene product may be involved in a system of error-prone repair.
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  • 55
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    Current genetics 6 (1982), S. 163-165 
    ISSN: 1432-0983
    Keywords: Hybridization ; Polyethylene glycol ; Nuclear transfer ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Viable hybrids of Saccharomyces cerevisiae were obtained by transfer of isolated diploid nuclei into haploid protoplasts using a polyethylene glycol (PEG) fusion procedure.
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  • 56
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    Current genetics 6 (1982), S. 159-162 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Transformation ; Gene subcloning
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    Topics: Biology
    Notes: Summary We have developped a procedure to delimit the boundaries of a cloned gene carried on a DNA fragment as large as 4 to 5 kilobases. The method consists in the following. Two series of limit digest products generated with a tetranucleotide recognition sequence endonuclease and originating from either of the two ends of this DNA segment are tested for their complementing capacity by yeast transformation. The gene is then delimited by the overlap of the two shortest complementing fragments.
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  • 57
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; TRP2 gene ; TRP3 gene ; Cloning in yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary This paper describes the isolation of the TRP2 and the TRP3 genes of Saccharomyces cerevisiae. Two pools of plasmids consisting of BamHI and Sa1GI yeast DNA inserts into the bifunctional yeast — Escherichia coli vector pLC544 (Kingsman et al. 1979) were constructed in E. coli and used for the isolation of the two genes by selection for functional complementation of trp2 and trp3 mutations, respectively, in yeast. The TRP2 gene was isolated on a 6.2 kb BamHl and a 5.8 kb Sa1GI yeast DNA fragment which shared an identical 4.5 kb BamHI-SaIGI fragment. The TRP3 gene was located on a 5.2 kb BamHl fragment. By physical, genetic and physiological experiments it could be shown that the cloned yeast DNA fragments contained the whole structural sequences as well as the regulatory regions of the TRP2 and the TRP3 genes.
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  • 58
    ISSN: 1432-072X
    Keywords: a Pheromone ; α Pheromone ; Hansenula wingei ; Inducible mutant ; Saccharomyces cerevisiae ; Saccharomyces kluyveri ; Sexual agglutinability ; Shmoo ; Synthetic analogues
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three analogues of the peptidyl pheromone, α pheromone of Saccharomyces kluyveri, synthesized based on the amino acid sequence proposed by Sato et al. (Agric Biol Chem 45:1531–1533, 1981) were tested for both shmoo-inducing and agglutinability-inducing actions. Purified natural α pheromone of the yeast showed the highest activity among the peptides tested. When methionine in the peptides was oxidized, the activity decreased significatly. α Pheromone of S. kluyveri induced sexual agglutinability in a cells of Saccharomyces cerevisiae, and shmoo in a cells of S. cerevisiae and S. kluyveri. a Pheromone of S. kluyveri had no agglutinability-inducing action on α cells of S. cerevisiae. a Cells of S. kluyveri inactivated only α pheromone of the same species, but a cells of S. cerevisiae inactivated α pheromones of both S. cerevisiae and S. kluyveri.
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  • 59
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    Archives of microbiology 132 (1982), S. 141-143 
    ISSN: 1432-072X
    Keywords: Phosphoenolpyruvate carboxykinase ; Gluconeogenesis ; Saccharomyces cerevisiae ; Mutant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A mutant of Saccharomyces cerevisiae lacking phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) was isolated. The mutant did not grow on gluconeogenic sources except glycerol. The mutation was recessive and apparently affected the structural gene of the enzyme. Intracellular levels of metabolites related to the metabolic situation of the enzyme were not significantly affected after transfer of the mutant from a medium with glycerol to a medium with ethanol as carbon source. In these conditions only AMP decreased 3 to 5 times. A search for mutants affected in the other gluconeogenic enzyme, fructose 1,6 bisphosphatase, remained unsuccessful.
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  • 60
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    Archives of microbiology 131 (1982), S. 298-301 
    ISSN: 1432-072X
    Keywords: Yeast ; Protoplast ; Compartmentation ; Vacuole ; Trehalose ; Trehalase ; Carbohydrate metabolism ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts of Saccharomyces cerevisiae synthesized and degraded trehalose when they were incubated in a medium containing traces of glucose and acetate. Such protoplasts were gently lyzed by the polybase method and a particulate and soluble fraction was prepared. Trehalose was found in the soluble fraction and the trehalase activity mostly in the particulate fraction which also contained the vacuoles besides other cell organelles. Upon purification of the vacuoles, by density gradient centrifugation, the specific activity of trehalase increased parallel to the specific content of vacuolar markers. This indicates that trehalose is located in the cytosol and trehalase in the vacuole. It is suggested that trehalose, in addition to its role as a reserve may also function as a protective agent to maintain the cytosolic structure under conditions of stress.
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  • 61
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    Archives of microbiology 133 (1982), S. 242-248 
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Tryptophan degradation to tryptophol ; Degradation-defective mutant strain ; Aromatic aminotransferases ; Tryptophan accumulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids. A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.
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  • 62
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    Archives of microbiology 132 (1982), S. 236-240 
    ISSN: 1432-072X
    Keywords: α Pheromone ; Cycloheximide ; Inducible a strain ; Phenylmethylsulfonyl fluoride ; Saccharomyces cerevisiae ; Sexual agglutinability ; Temperature-sensitive
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract When α pheromone-pretreated cells of an inducible a strain of Saccharomyces cerevisiae carrying the inducible gene saa1 were incubated in a growth medium at 28°C, induction of sexual agglutinability began after a 10 min lag period. If the cells were incubated at 38°C during the lag period, no induction occurred even after incubation at 28°C. Contrary to this, if the cells were incubated at 28°C during the lag period, almost complete induction occurred, even after transfer to 38°C. Temperature shift experiments revealed that 5 min incubation at 28°C was necessary for the initiation of the temperature-sensitive period and further 5 min incubation for the completion of the period. The temperature-sensitive period was sensitive to phenylmethylsulfonyl fluoride.
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  • 63
    ISSN: 1432-072X
    Keywords: Candida utilis ; Saccharomyces cerevisiae ; Zygosaccharomyces bailii ; Compartmentation ; Vacuoles ; Internal pH ; Phosphate ; Glycolysis ; Nuclear magnetic resonance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 31P NMR spectra were obtained from suspensions of Candida utilis, Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically on glucose. Direct introduction of substrate into the cell suspension, without interruption of the measurements, revealed rapid changes in pH upon addition of the energy source. All 31P NMR spectra of the yeasts studied indicated the presence of two major intracellular inorganic phosphate pools at different pH environments. The pool at the higher pH was assigned to cytoplasmic phosphate from its response to glucose addition and iodoacetate inhibition of glycolysis. After addition of substrate the pH in the compartment containing the second phosphate pool decreased. A parallel response was observed for a significant fraction of the terminal and penultimate phosphates of the polyphosphate observed by 31P NMR. This suggested that the inorganic phosphate fraction at the lower pH and the polyphosphates originated from the same intracellular compartment, most probably the vacuole. In this vacuolar compartment, pH is sensitive to metabolic conditions. In the presence of energy source a pH gradient as large as 0.8 to 1.5 units could be generated across the vacuolar membrane. Under certain conditions net transport of inorganic phosphate across the vacuolar membrane was observed during glycolysis: to the cytoplasm when the cytoplasmic phosphate concentration had become very low due to sugar phosphorylation, and into the vacuole when the former concentration had become high again after glucose exhaustion.
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  • 64
    ISSN: 1573-6881
    Keywords: Respiratory chain ; ATP synthesis ; mitochondria ; ubiquinone ; Saccharomyces cerevisiae ; cytochrome oxidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract Mitochondria, isolated from the ubiquinone-deficient nuclear mutant ofSaccharomyces cerevisiae E3-24, are practically unable to oxidize exogenous substrates. Respiratory activity, coupled to ATP synthesis, can, however, be reconstituted by the simple addition of ethanolic solutions of ubiquinones. A minimal length of the isoprenoid side chain (≥3) was required for the restoration. Saturation of the reconstitution required a large amount of exogeneous ubiquinone, in excess over the normal content present in the mitochondria of the wild type strain. A similar pattern of reconstituted activities could be also obtained using sonicated inverted particles. Mitochondria and sonicated particles are also able to carry out a dye-mediated electron flow coupled to ATP synthesis in the absence of added ubiquinone, using ascorbate or succinate as electron donor. This demonstrates that the energy conserving mechanism at the third coupling site of the respiratory chain is fully independent of the presence of the large mobile pool of ubiquinone in the membrane.
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  • 65
    ISSN: 1432-0983
    Keywords: Catalase ; Saccharomyces cerevisiae ; Heme ; Posttranscriptional control
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Compared to wild type cells, strains bearing the pleiotropic regulatory mutations cgr4 or cas1 synthesize apocatalase T at a high rate when grown on high glucose. Like heme-deficient ole3 single mutants, ole3 cgr4 and ole3 cas1 double mutants accumulate no catalase T protein in vivo. This defect introduced by the ole3 mutation is cured by the addition of ALA. By use of the inhibitor actinomycin D we confirm previous findings that ole3 mutants lack catalase T mRNA and show that (i) the ole3 cgr4 and ole3 cas1 double mutants do accumulate catalase T mRNA or mRNA precursor, and (ii) the processing or translation of this RNA or the accumulation of apocatalase T depends on the presence of home.
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  • 66
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    Current genetics 4 (1981), S. 85-90 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Nuclear isolation ; Percoll ; in vitro Transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A procedure has been developed for the rapid isolation of yeast nuclei in high yield using Percoll gradients. The nuclei are substantially free of cytoplasmic contamination as measured by alcohol dehydrogenase activities, have the typical chromatin digestion pattern when digested with nucleases, are useful for isolation of nuclear proteins and for in vitro transcription experiments.
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  • 67
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Yeast vectors ; Cosmids ; nif genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two vectors, termed pG63.11 (7.6 Kb) and pHCG3 (9.6 Kb), suitable for yeast transformation have been constructed. The pHCG3 vector has cosmid properties. Both vectors contain a single 3.3 Kb EcoRI-HindIII fragment of yeast origin which carries the yeast URA3 gene (1.1 Kb) and the origin of replication of the 2 µm plasmid (2.2 Kb). They confer ampicillin resistance and they contain 5 unique EcoRI,HpaI,HindIII,BamHI and SalI restriction sites. Cosmid pHCG3 was used to clone the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae carried by twoHindIII fragments of 17 and 26 Kb, respectively. The resulting cosmid, termed pGPC875 (53 Kb) which conferred a Nif+ phenotype to Escherichia coli, was introduced in yeast by transformation. No acetylene reduction activity was detectable in the transformants. However it was shown that the entire information for nitrogen fixation can be replicated and maintained intact in yeast for more than 50 generations of growth.
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  • 68
    ISSN: 1432-0983
    Keywords: Suppressors-tRNA ; Saccharomyces cerevisiae ; Nucleotide modification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have examined the tRNAs of two related strains of Saccharomyces cerevisiae, ψ + and ψ −, which differ with respect to an extrachromosomal genetic element that modulates the expression of genotypic and phenotypic suppression. Both the pattern of tRNAs synthesized and the level of nucleotide modification of several selected tRNA species were found to be the same in the ψ + and ψ − strains.
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  • 69
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    Current genetics 4 (1981), S. 135-143 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Mitochondria ; Gene cloning ; Transfer RNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have studied the organization and expression of a group of tRNA genes located on a 2,700 base pair portion of the yeast mitochondrial genome between the genetic markers cap (chloramphenicol resistance) and oxil (cytochrome oxidase subunit II). This region is spanned by mitochondrial DNA inserts of two recombinant plasmids, pYm162 and pYm267, which have been extensively mapped and sequenced. This tRNA group is composed of six tRNA genes, coding for tRNA AAR Lys , tRNA AGR Arg , tRNA GGN Gly , tRNA GAY Asp , tRNA AGY Ser , and tRNA CGN Arg . We report the sequence for the majority of the 2,700 base pair region including the genes for all six tRNAs. All six genes are oriented in the same direction and are, therefore, transcribed from the same DNA strand. Further, a comparison of the organization of this region with the analogous region of a related wild type strain shows that the tRNA gene order in the two strains is the same. Five of the six tRNA genes have corresponding transcripts in wild type RNA. Although a potential structural gene for tRNA CGN Arg is present, we do not detect a tRNA CGN Arg gene transcript.
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  • 70
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Borrelidin ; Aspartokinase ; Feedback regulation ; Threonine pool ; Homoserine pool ; S-Adenosylmethionine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Borrelidin is a specific inhibitor of the threonyl-tRNA-synthethase. A class of dominant borrelidin resistant mutants (BOR1) of Saccharomyces cerevisiae was biochemically characterized. The mutants possess an altered aspartokinase which is insensitive to threonine inhibition. The threonine and homoserine pools in these mutants are 22 times larger than in the wild type. By feeding aspartate under a variety of conditions the homoserine pool is increased even 57 times whilst the threonine pool is reduced.
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  • 71
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    Archives of microbiology 129 (1981), S. 146-149 
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Inhibition of tryptophanyl-tRNA synthetase ; Mode of action of tryptophan analogues ; Tryptophan analogue degradation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of the tryptophan analogue 4-methyltryptophan in Saccharomyces cerevisiae has been investigated. 4-Methyltryptophan inhibits the aminoacylation of tryptophan specific transfer ribonucleic acid (tRNATrp). The mode of inhibition is competitive and the analogue is not charged onto tRNATrp. Thus 4-methyltryptophan application depletes the cells from charged tRNATrp. As a consequence cell growth and protein synthesis are strongly reduced. 4-Methyltryptophan is degraded efficiently in culture media inoculated with the wild type strain; the effects of 4-methyltryptophan were therefore found to be transient.
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  • 72
    ISSN: 1432-072X
    Keywords: Fructose-1,6-bisphosphatase ; Phosphofructokinase ; Antagonistic enzymes ; Glycolytic intermediates ; ATP ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The intracellular levels of adenosine triphosphate and several glycolytic intermediates were determined in Saccharomyces cerevisiae in relation to the presence of the metabolically antagonistic enzymes phosphofructokinase and fructose-1,6-bisphosphatase. Phosphofructokinase is synthesized constitutively in cells grown in the presence of glucose and fructose-1,6-bisphosphatase derepression occurs upon the exhaustion of glucose from the growth medium. Transcriptional regulation of fructose-1,6-bisphosphatase was suggested based on experiments with wild type cells using 8-hydroxyquinoline, a known inhibitor of nuclear transcription, and with the S. cerevisiae mutant strain A364A (ts-136) blocked in the transport of nuclear RNA at non-permissive temperature. The level of phosphofructokinase was reduced more than 25-fold under conditions of high citrate accumulation in an aconitaseless, glutamate requiring mutant strain, MO-1-9B. There was a rapid decrease in the levels of adenosine triphosphate and fructose-1,6-bisphosphate at the end of log-phase of culture growth when both fructose-1,6-bisphosphatase and phosphofructokinase were present in the cells simultaneously. The changes in the levels of key glycolytic intermediates, but not the changes in adenosine triphosphate, during the simultaneous presence of these two enzymes, can be explained without involving any futile cycling.
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  • 73
    ISSN: 1432-072X
    Keywords: Hansenula wingei ; Induction ; Saccharomyces cerevisiae ; Saccharomyces kluyveri ; Sex pheromone ; Sexual agglutinability ; Sexual agglutination
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast, Hansenula wingei has two mating types designated 5 and 21. Cells of each mating type were found to produce mating type-specific sex pheromone which induces sexual agglutinability of the opposite mating type. Crude fractions of these pheromones were prepared by using an Amberlite CG 50 (H+ type) column. The agglutinability-inducing action of the pheromones required glucose as carbon source, but no external nitrogen source. The action of the pheromones was inhibited by 5 μg/ml cycloheximide. The optimum pH for the pheromone action was 4.0. α Pheromones of Saccharomyces cerevisiae and Saccharomyces kluyveri induced sexual agglutinability of 5 mating type cells but did not that of 21 mating type cells. a Pheromones of the Saccharomyces yeasts had no effect on both 5 and 21 mating type cells. The sex pheromones of H. wingei had no effect on the sexual agglutinability of inducible a cells of S. cerevisiae. From the experimental results obtained so far, we propose to call 5 and 21 mating types in H. wingei a and α mating types, respectively.
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  • 74
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Bud scar ; Scar ring ; Glucan ; Chitin ; Mannan ; Cytology ; Electron and X-ray diffractions ; Physico-chemical characterization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Scar rings (SR) from scarless cells at the early stages of budding and mature bud scars from Saccharomyces cerevisiae isolated by both chemical and enzymic treatment of cell walls were observed by selected-area electron diffraction (SAED), X-ray diffraction and electron microscopy with simultaneous physico-chemical characterization (including molar mass, intrinsic viscosity and crystallite size) of α-chitin and glucan. The SR, composed of glucan with strong 0.608; 0.397 and 0.293 nm X-ray reflections, was formed at the start of budding. The SAED patterns of α-chitin both in the adjacent circular zone and in the parts of newly formed primary septum (PS), observed when the development of the PS started, did not differ from those of α-chitin in the single mature bud scar. The bud scar consisted of α-chitin, glucan and mannan and their content, as well as the crystallite size of chitin, depended on the mode of preparation.
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  • 75
    ISSN: 1432-0983
    Keywords: Gene cloning ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have carried out experiments aimed at explaining the observed variations in transformation frequencies when Saccharomyces cerevisiae or Saccharomyces carlbergensis are transformed with chimeric plasmids that contain one of 4 possible EcoRI fragments of the yeast 2-μm circle. These plasmids fall into 2 classes when used to transform 2 different yeast his3 auxotrophs, one (strain LL20) harbours indigenous 2-μm circle, and the other (strain YF233) is devoid of this plasmid. Hybrid plasmids containing either the 2.4 mega-dalton (mD) R-form EcoRI fragment (pYF88) or the l.4 mD L-form EcoRI fragment (pYF177) of 2-μm circle transform either of the two hosts at a high frequency (50,000 colonies per Mg in LL20 and 10,000 colonies per μg in YF233). Hybrid plasmids containing the 1.5 mD R-form EcoRI fragment (pYF87) or the 2.5 mD L-form EcoRI fragment (pYF178) of the 2-μm circle transform LL20 at a reduced frequency (6,000–16,000 colonies per μg) and YF233 at extremely low frequencies (1–5 colonies per μg). All plasmids retrieved from strain YF233 that had been transformed with pYF88 or pYF177 were identical to the original transforming plasmid. Of the plasmids retrieved from strain LL20 that had been transformed with pYF87 and pYF178, approximately half had acquired an extra copy of the 2-μm circle. Of the plasmids retrieved from strain LL20 that had been transformed with pYF88 and pYF177, an average of only approximately 13% had acquired an extra copy of 2-μm circle. Taken together, these observations indicate that the transformation of yeast by a plasmid lacking the ability to replicate (pYF87 and pYF1780) occurs by the recombinational acquisition of 1 copy of the host 2-μm circle, which serves to supply the incoming plasmid with missing essential sequences. A comparison of 2-μm circle DNA fragments carried by pYF88 and pYF177 indicates that the region of 2-μm circle required for high frequency transformation is a 1.2 mD segment that is common to the 2.4 mD R-form and 1.4 ml) L-form EcoRI fragments. This region extends from the EcoRI cut site adjacent to the PstI site, through to the end of the inverted repeat. However, the inverted repeat sequence alone is not sufficient to bestow high frequency transformation of yeast.
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  • 76
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    Current genetics 2 (1980), S. 115-120 
    ISSN: 1432-0983
    Keywords: Galactose fermentation ; Saccharomyces cerevisiae ; Regulatory mutant
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: 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.
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    Current genetics 2 (1980), S. 223-228 
    ISSN: 1432-0983
    Keywords: Transcriptional Units ; GAL Genes ; Saccharomyces cerevisiae ; UV mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The size of the transcriptional unit of the structural genes for three galactose-metabolizing enzymes which form a cluster on chromosome II in Saccharomyces cerevisiae was studied by the ultraviolet light (UV)-mapping technique. Thus the size of the primary transcripts of GAL7 for galactose-1-phosphate uridylyl transferase, GAL10 for uridine diphosphoglucose 4-epimerase, or GAL1 for galactokinase were estimated to be 0.81 x 106, 1.1 x 106, or 1.3 x 106 respectively. In the light of these data together with the known directions of transcription of the genes, we concluded that each of three genes was transcribed from its own promoter.
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  • 78
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    Archives of microbiology 128 (1980), S. 157-161 
    ISSN: 1432-072X
    Keywords: Lactobacillus brevis ; Streptococcus lactis ; Saccharomyces cerevisiae ; Concanavalin A ; Symbiosis ; Tibi
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Tibi grains consist of a unique and very stable symbiotic association of Lactobacillus brevis, Streptococcus lactis and Saccharomyces cerevisiae embedded in a dextran matrix. The structural organization of the grain was examined by light, scanning and transmission electron microscopy. The grain was constituted of an outer compact layer and an inner spongy structure. The outer layer was more densely populated by the microorganisms than the inner layer but dextran, stained on frozen thin sections with fluorescein-conjugated concanavalin A, was more abundant in the inner layer.
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  • 79
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    Archives of microbiology 125 (1980), S. 89-95 
    ISSN: 1432-072X
    Keywords: Sulfur dioxide ; Sulfite ; Air polluting substances ; Saccharomyces cerevisiae ; Active agent of irreversible cell inactivation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract After a short period of tolerance, living cells of Saccharomyces cerevisiae were irreversibly damaged by low concentrations of sulfite. The length of the period of tolerance and the rate of the damaging effect depended on the concentration on sulfite, pH-value, temperature, the physiological state of the cells, and incubation time. Inhibitors of protein synthesis and mitochondrial ATP synthesis did not alter the deleterious effect of sulfite on living cells. Furthermore, cell damage leading to inhibition of colony formation occured under aerobic as well as under anaerobic conditions. Prior to cell inactivation sulfite induced the formation of respiratory deficient cells. The active agent was shown to be SO2.
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  • 80
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Catabolite repression and inactivation ; Inhibition of protease B
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Catabolite inactivation of fructose-1,6-bisphosphatase, isocitrate lyase, phosphoenolpruvate carboxykinase and malate dehydrogenase in intact cells could be prevented by phenylmethylsulfonyl fluoride added 40 min prior to the addition of glucose. Protein synthesis, fermentative and respiratory activity and catabolite repression were not affected. Elimination of catabolite inactivation by the addition of PMSF revealed that catabolite repression started at different times for different enzyme.
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  • 81
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    Archives of microbiology 124 (1980), S. 285-287 
    ISSN: 1432-072X
    Keywords: Yeast ; Saccharomyces cerevisiae ; Heat killing ; Membrane damage ; Genetic damage ; Growth temperature
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The resistance of exponentially growing yeast cells to killing by exposure to 52°C increased markedly as the growth temperature was increased. Identical killing curves were obtained for cells suspended in growth medium or in 0.9% saline. Cells resistant to killing at 52°C were quite sensitive to killing at slightly higher temperatures. These results suggest a primary role for membrane damage in the mechanism of heat killing.
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  • 82
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    Plant and soil 57 (1980), S. 41-47 
    ISSN: 1573-5036
    Keywords: Amino acids ; Nucleic acid bases ; Saccharomyces cerevisiae ; Vitamins ; Yeast autolyzate ; Yield stimulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Effect of yeast autolyzate on the growth of vineless pea plant was investigated. The yield was increased remarkably when yeast autolyzate was added, as compared with the control area in which the plants received only mineral fertilizer solution. The yield stimulation was the highest where 0.1 ppm of the autolyzate was added, the yield increasing by more than 80%. The reason why the yields were lower in the areas where the amount of yeast autolyzate added was incrased to 1 ppm and to 10 ppm, was presumed to be due to the fact that the substances may have been absorbed, decomposed, and utilized by soil microorganisms.
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