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  • Articles  (2,155)
  • Photosystem II  (1,079)
  • Saccharomyces cerevisiae  (1,076)
  • Elsevier  (1,170)
  • Springer  (985)
  • American Institute of Physics (AIP)
  • Molecular Diversity Preservation International
  • 1
    Electronic Resource
    Electronic Resource
    A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae (2000)
    Springer
    BioMetals 13 (2000), S. 65-72 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009250017785
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  • 2
    Electronic Resource
    Electronic Resource
    A screen of yeast respiratory mutants for sensitivity against the mycotoxin citrinin identifies the vacuolar ATPase as an essential factor for the toxicity mechanism (2000)
    Springer
    Current genetics 37 (2000), S. 227-284 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002940070001
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  • 3
    Electronic Resource
    Electronic Resource
    POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase δ, defines a link between DNA replication and the mutagenic bypass repair pathway (2000)
    Springer
    Current genetics 38 (2000), S. 178-187 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002940000149
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  • 4
    Electronic Resource
    Electronic Resource
    A two-dimensional many-body system with competing interactions as a model for segregation of photosystems in thylakoids of green plants (2000)
    Springer
    European biophysics journal 29 (2000), S. 214-220 
    Details
    ISSN: 1432-1017
    Keywords: Key words Lipid-protein interactions ; Photosystem I ; Photosystem II ; Thylakoid membranes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract  We address the segregation of photosystems I (PSI) and II (PSII) in thylakoid membranes by means of a molecular dynamics method. We assume a two-dimensional (in-plane) problem with PSI and PSII being represented by particles with different values of negative charge. The pair interactions between particles include a screened Coulomb repulsive part and am exponentially decaying attractive part. Our modeling results suggest that the system may have a complicated phase behavior, including a quasi-crystalline phase at low ionic screening, a disordered phase and, in addition, a possible “clotting” agglomerate phase at high screening where the photosystems tend to clot together. The relevance of the observed phenomena to the stacking of thylakoid membranes is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002490000080
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  • 5
    Electronic Resource
    Electronic Resource
    Endopolygalacturonase genes and enzymes from Saccharomyces cerevisiae and Kluyveromyces marxianus (2000)
    Springer
    Current genetics 38 (2000), S. 264-270 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002940000160
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  • 6
    Electronic Resource
    Electronic Resource
    Recessive mutations in SUP35 and SUP45 genes coding for translation release factors affect chromosome stability in Saccharomyces cerevisiae (2000)
    Springer
    Current genetics 37 (2000), S. 285-291 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002940050529
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  • 7
    Electronic Resource
    Electronic Resource
    Interaction of nitric oxide with the oxygen evolving complex of photosystem II and manganese catalase: a comparative study (2000)
    Springer
    JBIC 5 (2000), S. 354-363 
    Details
    ISSN: 1432-1327
    Keywords: Catalase ; Manganese cluster ; Nitric oxide ; Photosystem II
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Thermus thermophilus catalase. Flash fluorescence studies indicate that the S3 state of the OEC in the presence of ca. 0.6 mM NO is reduced to the S1 with an apparent halftime of ca. 0.4 s at about 18 °C, compared with a biphasic decay, with approximate halftimes of 28 s for S3 to S2 and 140 s for S2 to S1 in the absence of NO. Under similar conditions the S2 state is reduced by NO to the S1 state with an approximate halftime of 2 s. These results extend a recent study indicating a slow reduction of the S1 state at −30°C, via the S0 and S−1 states, to a Mn(II)-Mn(III) state resembling the corresponding state in catalase. The reductive mode of action of NO is repeated with the di-Mn cluster of catalase: the Mn(III)-Mn(III) redox state is reduced to the Mn(II)-Mn(II) state via the intermediate Mn(II)-Mn(III) state. The kinetics of this reduction suggest a decreasing reduction potential with decreasing oxidation state, similar to what is observed with the active states of the OEC. What is unique about the OEC is the rapid interaction of NO with the S3 state of the OEC, which is compatible with a metalloradical character of this state.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00010664
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  • 8
    Electronic Resource
    Electronic Resource
    Yeast Intracellular Water Determination by Thermogravimetry (2000)
    Springer
    Journal of thermal analysis and calorimetry 59 (2000), S. 643-648 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1010172830355
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  • 9
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    Molecular Modeling of the Complexes between Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase and the ATP Analogs Pyridoxal 5′-Diphosphoadenosine and Pyridoxal 5′-Triphosphoadenosine. Specific Labeling of Lysine 290 (2000)
    Springer
    The protein journal 19 (2000), S. 67-73 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007099010762
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  • 10
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    Involvement of the Inverted Repeat of the yeast 2-micron plasmid in Flp site-specific and RAD52-dependent homologous recombination (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 81-89 
    Details
    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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    URL: http://dx.doi.org/10.1007/PL00008678
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  • 11
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    Ambient pH signalling in ascomycetous yeasts involves homologues of theAspergillus nidulans genes palF and palH (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 505-513 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380051195
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  • 12
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    Multiple copies of MRG19 suppress transcription of the GAL1 promoter in a GAL80 -dependent manner in Saccharomyces cerevisiae (2000)
    Springer
    Molecular genetics and genomics 262 (2000), S. 1113-1122 
    Details
    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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    URL: http://dx.doi.org/10.1007/PL00008654
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  • 13
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    Electronic Resource
    Structure-function relationships in replication origins of the yeast Saccharomyces cerevisiae: higher-order structural organization of DNA in regions flanking the ARS consensus sequence (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 854-866 
    Details
    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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380000253
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  • 14
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    Characterization of staurosporine-sensitive mutants of Saccharomyces cerevisiae: vacuolar functions affect staurosporine sensitivity (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 877-888 
    Details
    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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    URL: http://dx.doi.org/10.1007/s004380000255
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  • 15
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    MUS81 encodes a novel Helix-hairpin-Helix protein involved in the response to UV- and methylation-induced DNA damage in Saccharomyces cerevisiae (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 812-827 
    Details
    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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    URL: http://dx.doi.org/10.1007/s004380000241
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  • 16
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    Functional characterization of the PS II–LHC II supercomplex isolated by a direct method from spinach thylakoid membranes (2000)
    Springer
    Photosynthesis research 64 (2000), S. 179-187 
    Details
    ISSN: 1573-5079
    Keywords: EPR ; fluorescence ; Photosystem II ; thermoluminescence ; thylakoid membranes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Recently, a novel procedure to isolate a highly pure and active Photosystem II preparation directly from thylakoid membranes, referred to as PS II–LHC II supercomplex, was reported [Eshaghi et al. (1999) FEBS Lett 446: 23–26]. In addition to the reaction center core proteins, the supercomplex contains all the extrinsic proteins of the oxygen evolving complex and a set of chlorophyll a/b binding proteins. In this paper, the functional properties of this isolated supercomplex are further characterized by using EPR spectroscopy, thermoluminescence, fluorescence relaxation kinetics and flash induced oxygen yield measurements. The PS II–LHC II supercomplex contains, in addition to QA and QB, a small pool of plastoquinone (PQ). Although the isolated complex is no longer membrane bound, it has preserved functional characteristics of a well defined PS II preparation with the exception of some modification of QB sites.
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    URL: http://dx.doi.org/10.1023/A:1006404302573
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  • 17
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    Solubilization of green plant thylakoid membranes with n-dodecyl-α,D-maltoside. Implications for the structural organization of the Photosystem II, Photosystem I, ATP synthase and cytochrome b6 f complexes (2000)
    Springer
    Photosynthesis research 64 (2000), S. 155-166 
    Details
    ISSN: 1573-5079
    Keywords: CF0F1 ; cytochrome b 6 f ; electron microscopy ; grana ; Photosystem I ; Photosystem II
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A biochemical and structural analysis is presented of fractions that were obtained by a quick and mild solubilization of thylakoid membranes from spinach with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by a partial purification using gel filtration chromatography. The largest fractions consisted of paired, appressed membrane fragments with an average diameter of about 360 nm and contain Photosystem II (PS II) and its associated light-harvesting antenna (LHC II), but virtually no Photosystem I, ATP synthase and cytochrome b 6 f complex. Some of the membranes show a semi-regular ordering of PS II in rows at an average distance of about 26.3 nm, and from a partially disrupted grana membrane fragment we show that the supercomplexes of PS II and LHC II represent the basic structural unit of PS II in the grana membranes. The numbers of free LHC II and PS II core complexes were very high and very low, respectively. The other macromolecular complexes of the thylakoid membrane occurred almost exclusively in dispersed forms. Photosystem I was observed in monomeric or multimeric PS I-200 complexes and there are no indications for free LHC I complexes. An extensive analysis by electron microscopy and image analysis of the CF0F1 ATP synthase complex suggests locations of the δ (on top of the F1 headpiece) and ∈ subunits (in the central stalk) and reveals that in a substantial part of the complexes the F1 headpiece is bended considerably from the central stalk. This kinking is very likely not an artefact of the isolation procedure and may represent the complex in its inactive, oxidized form.
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    URL: http://dx.doi.org/10.1023/A:1006476213540
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  • 18
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    Selective replacement of the active and inactive pheophytin in reaction centres of Photosystem II by 131-deoxo-131-hydroxy-pheophytin a and comparison of their 6 K absorption spectra (2000)
    Springer
    Photosynthesis research 64 (2000), S. 189-198 
    Details
    ISSN: 1573-5079
    Keywords: Photosystem II ; pheophytin a ; pigment exchange ; reaction centre ; 131-deoxo-131-hydroxy pheophytin a
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pheophytin a (Pheo) in Photosystem II reaction centres was exchanged for 131-deoxo-131-hydroxy-pheophytin a (131-OH-Pheo). The absorption bands of 131-OH-Pheo are blue-shifted and well separated from those of Pheo. Two kinds of modified reaction centre preparations can be obtained by applying the exchange procedure once (RC1×) or twice (RC2×). HPLC analysis and Pheo QX absorption at 543 nm show that in RC1× about 50% of Pheo is replaced and in RC2× about 75%. Otherwise, the pigment and protein composition are not modified. Fluorescence emission and excitation spectra show quantitative excitation transfer from the new pigment to the emitting chlorophylls. Photoaccumulation of Pheo− is unmodified in RC1× and decreased only in RC2×, suggesting that the first exchange replaces the inactive and the second the active Pheo. Comparing the effects of the first and the second replacement on the absorption spectrum at 6 K did not reveal substantial spectral differences between the active and inactive Pheo. In both cases, the absorption changes in the QY region can be interpreted as a combination of a blue shift of a transition at 684 nm, a partial decoupling of chlorophylls absorbing at 680 nm and a disappearance of Pheo absorption in the 676-680 nm region. No absorption decrease is observed at 670 nm for RC1× or RC2×, showing that neither of the two reaction centre pheophytins contributes substantially to the absorption at this wavelength.
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    URL: http://dx.doi.org/10.1023/A:1006407314449
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    Excitation energy transfer in aggregates of Photosystem I and Photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: Can assembly of the pigment-protein complexes control the extent of spillover? (2000)
    Springer
    Photosynthesis research 64 (2000), S. 199-207 
    Details
    ISSN: 1573-5079
    Keywords: cyanobacteria ; excitation energy transfer ; membrane proteins assembly ; Photosystem I ; Photosystem II ; spillover ; state transitions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The fluorescence profile of Photosystem I/Photosystem II mixtures in different solvent systems shows that both non-hydrophobic and hydrophobic interactions govern their association and control energy transfer from Photosystem II to Photosystem I. The non-hydrophobic interactions lead to a highly efficient excitation energy transfer from Photosystem II to Photosystem I. In view of this, we propose that similar non-hydrophobic interactions, between the Photosystem II and Photosystem I peripheral proteins, also play a significant role in their association in thylakoids that control state transitions in cyanobacteria.
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    URL: http://dx.doi.org/10.1023/A:1006485823403
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    Light-induced quenching of chlorophyll fluorescence at 77 K in leaves, chloroplasts and Photosystem II particles (2000)
    Springer
    Photosynthesis research 65 (2000), S. 219-229 
    Details
    ISSN: 1573-5079
    Keywords: energy dissipation ; Photosystem I ; Photosystem II ; spectroscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The light-induced chlorophyll (Chl) fluorescence decline at 77 K was investigated in segments of leaves, isolated thylakoids or Photosystem (PS) II particles. The intensity of chlorophyll fluorescence declines by about 40% upon 16 min of irradiation with 1000 μmol m−2 s−1 of white light. The decline follows biphasic kinetics, which can be fitted by two exponentials with amplitudes of approximately 20 and 22% and decay times of 0.42 and 4.6 min, respectively. The decline is stable at 77 K, however, it is reversed by warming of samples up to 270 K. This proves that the decline is caused by quenching of fluorescence and not by pigment photodegradation. The quantum yield for the induction of the fluorescence decline is by four to five orders lower than the quantum yield of QA reduction. Fluorescence quenching is only slightly affected by addition of ferricyanide or dithionite which are known to prevent or stimulate the light-induced accumulation of reduced pheophytin (Pheo). The normalised spectrum of the fluorescence quenching has two maxima at 685 and 695 nm for PS II emission and a plateau for PS I emission showing that the major quenching occurs within PS II. ‘Light-minus-dark’ difference absorbance spectra in the blue spectral region show an electrochromic shift for all samples. No absorbance change indicating Chl oxidation or Pheo reduction is observed in the blue (410–600 nm) and near infrared (730–900 nm) spectral regions. Absorbance change in the red spectral region shows a broad-band decrease at approximately 680 nm for thylakoids or two narrow bands at 677 and 670–672 nm for PS II particles, likely resulting also from electrochromism. These absorbance changes follow the slow component of the fluorescence decline. No absorbance changes corresponding to the fast component are found between 410 and 900 nm. This proves that the two components of the fluorescence decline reflect the formation of two different quenchers. The slow component of the light-induced fluorescence decline at 77 K is related to charge accumulation on a non-pigment molecule of the PS II complex.
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    URL: http://dx.doi.org/10.1023/A:1010730103382
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    Low-frequency fourier transform infrared spectroscopy of the oxygen-evolving complex in Photosystem II* (2000)
    Springer
    Photosynthesis research 66 (2000), S. 57-63 
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    ISSN: 1573-5079
    Keywords: FTIR ; manganese cluster ; oxygen-evolving complex ; Photosystem II ; plastoquinone ; vibrational spectroscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this communication, we report our progress on the development of low-frequency Fourier transform infrared (FTIR) spectroscopic techniques to study metal-substrate and metal-ligand vibrational modes in the Photosystem II/oxygen-evolving complex (PS II/OEC). This information will provide important structural and mechanistic insight into the OEC. Strong water absorption in the low-frequency region (below 1000 cm−1), a lack of suitable materials, and temperature control problems have limited previous FTIR spectroscopic studies of the OEC to higher frequencies (〉1000 cm−1). We have overcome these technical difficulties that have blocked access to the low-frequency region and have developed successive instruments that allow us to move deeper into the low-frequency region (down to 350 cm−1), while increasing both data accumulation efficiency and S/N ratio. We have detected several low-frequency modes in the S2/S1spectrum that are specifically associated with these two states. Our results demonstrate the utility of FTIR techniques in accessing low-frequency modes in Photosystem II and in proteins generally.
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    URL: http://dx.doi.org/10.1023/A:1010779327960
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    Airborne test of laser pump-and-probe technique for assessment of phytoplankton photochemical characteristics (2000)
    Springer
    Photosynthesis research 66 (2000), S. 45-56 
    Details
    ISSN: 1573-5079
    Keywords: chlorophyll ; energy quenching ; fluorescence ; LIDAR ; photochemistry ; photosynthesis ; Photosystem II ; phytoplankton ; pump and probe ; remote sensing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Initial results of the airborne LIDAR measurement of photochemical quantum yield, ΦPo, and functional absorption cross-section, σPS II, of Photosystem II (PS II) are reported. NASA's AOL3 LIDAR was modified to implement short-pulse pump-and-probe (SP-P&P) LIDAR measurement protocol. The prototype system is capable of measuring a pump-induced increase in probe-stimulated chlorophyll fluorescence, ΔF/Fsat, along with the acquisition of `conventional' LIDAR-fluorosensor products from an operational altitude of 150 m. The use of a PS II sub-saturating probe pulse increases the response signal but also results in excessive energy quenching (EEQ) affecting the ΔF/Fsat magnitude. The airborne data indicated up to a 3-fold EEQ-caused decline in ΔF/Fsat, and 2-fold variability in the EEQ rate constant over a spatial scale a few hundred kilometers. Therefore, continuous monitoring of EEQ parameters must be incorporated in the operational SP-P&P protocol to provide data correction for the EEQ effect. Simultaneous airborne LIDAR measurements of ΦPo and σPS II with EEQ correction were shown to be feasible and optimal laser excitation parameters were determined. Strong daytime ΔF/Fsat decline under ambient light was found in the near-surface water layer over large aquatic areas. An example of SP-P&P LIDAR measurement of phytoplankton photochemical and fluorescent characteristics in the Chesapeake Bay mouth is presented. Prospects for future SP-P&P development and related problems are discussed.
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    URL: http://dx.doi.org/10.1023/A:1010764420934
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    An FTIR study on the structure of the oxygen-evolving Mn-cluster of Photosystem II in different spin forms of the S2 state (2000)
    Springer
    Photosynthesis research 63 (2000), S. 47-57 
    Details
    ISSN: 1573-5079
    Keywords: Fourier transform infrared spectroscopy ; Mn-cluster ; oxygen evolution ; photosynthesis ; Photosystem II ; vibrational spectroscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The S2 state of the oxygen-evolving Mn-cluster of Photosystem II (PS II) is known to have different forms that exhibit the g =2 multiline and g = 4.1 EPR signals. These two spin forms are interconvertible at 〉 200 K and the relative amplitudes of the two signals are dependent on the species of cryoprotectant and alcohol contained in the medium. Also, it was recently found that the mutiline form can be converted to the g = 4.1 form by absorption of near-infrared light by the Mn-cluster itself at around 150 K [Boussac et al. (1996) Biochemistry 35: 6984–6989]. We have used light-induced Fourier transform infrared (FTIR) difference spectroscopy to study the structural difference in these two S2 forms. FTIR difference spectra for S2/S1 as well as for S2QA -/S1QA measured at cryogenic temperatures using PS II membranes in the presence of various cryoprotectants, and monohydric alcohols did not show any specific differences except for intensities of amide I bands, which were larger when ethylene glycol or glycerol was present in addition to sucrose. This result was interpreted due to more flexible movement of the protein backbones upon S2 formation with a higher cryoprotectant content. Light-induced difference spectra measured at 150 K using either blue light without near-infrared light or red plus near-infrared light also did not show any detectable difference. In addition, a different spectrum upon near-infrared illumination at 150 K of the PS II sample in which the S2 state had been photogenerated at 200 K exhibited no meaningful signals. These results indicate that the two S2 forms that give rise to the multiline and g = 4.1 signals have only minor differences, if any, in the structures of amino-acid ligands and polypeptide backbones. This conclusion suggests that conversion between the two spin states is caused by a spin-state transition in the Mn(III) ion rather than valence swapping within the Mn-cluster that would considerably affect the vibrations of ligands.
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    URL: http://dx.doi.org/10.1023/A:1006362118267
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    Short-pulse pump-and-probe technique for airborne laser assessment of Photosystem II photochemical characteristics (2000)
    Springer
    Photosynthesis research 66 (2000), S. 33-44 
    Details
    ISSN: 1573-5079
    Keywords: chlorophyll ; fluorescence ; LIDAR ; photosynthesis ; Photosystem II ; pump and probe ; remote sensing ; singlet-singlet quenching ; singlet-triplet quenching
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The development of a technique for laser measurement of fPhotosystem II (PS II) photochemical characteristics of phytoplankton and terrestrial vegetation from an airborne platform is described. Results of theoretical analysis and experimental study of pump-and-probe measurement of the PS II functional absorption cross-section and photochemical quantum yield are presented. The use of 10 ns probe pulses of PS II sub-saturating intensity provides a significant, up to 150-fold, increase in the fluorescence signal compared to conventional `weak-probe' protocol. Little effect on the fluorescence yield from the probe-induced closure of PS II reaction centers is expected over the short pulse duration, and thus a relatively intense probe pulse can be used. On the other hand, a correction must be made for the probe-induced carotenoid triplet quenching and singlet-singlet annihilation. A Stern-Volmer model developed for this correction assumes a linear dependence of the quenching rate on the laser pulse fluence, which was experimentally validated. The PS II saturating pump pulse fluence (532 nm excitation) was found to be 10 and 40 μmol quanta m−2 for phytoplankton samples and leaves of higher plants, respectively. Thirty μs was determined as the optimal delay in the pump-probe pair. Our results indicate that the short-pulse pump-and-probe measurement of PS II photochemical characteristics can be implemented from an airborne platform using existing laser and LIDAR technologies.
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    URL: http://dx.doi.org/10.1023/A:1010795820025
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    Low resistance against novel 2-benzylamino-1,3,5-triazine herbicides in atrazine-resistant Chenopodium album plants (2000)
    Springer
    Photosynthesis research 65 (2000), S. 115-120 
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    ISSN: 1573-5079
    Keywords: electron transport ; herbicides ; novel triazines ; O-J-I-P fluorescence transient ; Photosystem II ; resistance ; thylakoid membrane
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effects of nine novel 2-benzylamino-1,3,5-triazines on photosynthetic reactions were measured in thylakoids isolated from wild-type and atrazine-resistant plants of Chenopodium album. The resistant plants have a mutation of serine for glycine at position 264 of the D1 protein. The measurement of oxygen evolution and chlorophyll a fluorescence induction indicated a 2–4-fold stronger inhibition by the 6-trifluoromethyl analogues of Photosystem II-dependent electron flow than atrazine. Analogues having a 6-methyl-, 6-monofluoromethyl or 6-difluoromethyl substitution were weak inhibitors, indicating that the 6-trifluoro group is very important for strong inhibition. All the nine novel 2-benzylamino-1,3,5-triazines were almost as active in wild-type as in atrazine-resistant thylakoids, indicating that the benzylamino substitution may be important for the lack of resistance in the atrazine-resistant plants.
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    URL: http://dx.doi.org/10.1023/A:1006493703939
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    Nitrogen ligation to the manganese cluster of Photosystem II in the absence of the extrinsic proteins and as a function of pH (2000)
    Springer
    Photosynthesis research 65 (2000), S. 175-185 
    Details
    ISSN: 1573-5079
    Keywords: electron paramagnetic resonance ; extrinsic proteins ; manganese cluster ; oxygen evolution ; photosynthesis ; Photosystem II
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three extrinsic proteins (PsbO, PsbP and PsbQ), with apparent molecular weights of 33, 23 and 17 kDa, bind to the lumenal side of Photosystem II (PS II) and stabilize the manganese, calcium and chloride cofactors of the oxygen evolving complex (OEC). The effect of these proteins on the structure of the tetramanganese cluster, especially their possible involvement in manganese ligation, is investigated in this study by measuring the reported histidine-manganese coupling [Tang et al. (1994) Proc Natl Acad Sci USA 91: 704–708] of PS II membranes depleted of none, two or three of these proteins using ESEEM (electron spin echo envelope modulation) spectroscopy. The results show that neither of the three proteins influence the histidine ligation of manganese. From this, the conserved histidine of the 23 kDa protein can be ruled out as a manganese ligand. Whereas the 33 and 17 kDa proteins lack conserved histidines, the existence of a 33 kDa protein-derived carboxylate ligand has been posited; our results show no evidence for a change of the manganese co-ordination upon removal of this protein. Studies of the pH-dependence of the histidine–manganese coupling show that the histidine ligation is present in PS II centers showing the S2 multiline EPR signal in the pH-range 4.2–9.5.
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    URL: http://dx.doi.org/10.1023/A:1006435432185
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    Degradation of the Photosystem II D1 and D2 proteins in different strains of the cyanobacterium Synechocystis PCC 6803 varying with respect to the type and level of psbA transcript (2000)
    Springer
    Plant molecular biology 42 (2000), S. 635-645 
    Details
    ISSN: 1573-5028
    Keywords: cyanobacteria ; D1 ; D2 ; Photosystem II ; psbA ; Synechocystis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The turnover of the D1 and D2 proteins of Photosystem II (PSII) has been investigated by pulse-chase radiolabeling in several strains of the cyanobacterium Synechocystis PCC 6803 containing different types and levels of the psbA transcript. Strains lacking psbA1 and psbA3 gene and containing high levels of the psbA2 transcript showed the selective synthesis of D1 whose degradation could be slowed down by the protein synthesis inhibitor lincomycin. In contrast, in strains containing just the psbA3 gene, the intensity of the D1 protein labeling was lower and labeling of the D2 and CP43 proteins was stimulated in comparison to the psbA2-containing strains. In addition, the rate and selectivity of the D1 degradation and its dependence on the presence of lincomycin was proportional to the level of the psbA3 transcript in the particular strain. Consequently, there was parallel, lincomycin-independent and slowed-down breakdown of the D1 and D2 proteins in strains with the lowest level of psbA3 transcript. These results are discussed in terms of a model in which the rate of D1 and D2 degradation in cyanobacteria is affected not only by the rate of PSII photodamage, but also by the availability of newly synthesized D1 protein. Moreover, the comparison of the non-oxygen-evolving D1 mutants D170A** and Y161F*** differing by the presence of tyrosine Z has indicated a minor role of the oxidized form of this secondary PSII electron donor in the donor side mechanism of D1 and D2 protein breakdown.
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    URL: http://dx.doi.org/10.1023/A:1006305308196
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    Partial Assembly of the Yeast Mitochondrial ATP Synthase 1 (2000)
    Springer
    Journal of bioenergetics and biomembranes 32 (2000), S. 391-400 
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    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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    URL: http://dx.doi.org/10.1023/A:1005532104617
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    Electron microscopy of the K2 killer effect of Saccharomyces cerevisiae T206 on a mesophilic wine yeast (2000)
    Springer
    Antonie van Leeuwenhoek 78 (2000), S. 117-122 
    Details
    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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    URL: http://dx.doi.org/10.1023/A:1026588220367
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    Pseudohyphal growth is induced in Saccharomyces cerevisiae by a combination of stress and cAMP signalling (2000)
    Springer
    Antonie van Leeuwenhoek 78 (2000), S. 187-194 
    Details
    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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    URL: http://dx.doi.org/10.1023/A:1026594407609
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    Electron spin-lattice relaxation studies of different forms of the S2 state multiline EPR signal of the Photosystem II oxygen-evolving complex (2000)
    Springer
    Photosynthesis research 66 (2000), S. 189-198 
    Details
    ISSN: 1573-5079
    Keywords: electron spin-lattice relaxation rate ; manganese ; oxygen-evolving complex ; photosynthesis ; Photosystem II
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The pulsed EPR inversion recovery sequence has been utilized to monitor the temperature dependence of the electron spin-lattice relaxation rate of the Mn cluster of the Photosystem II oxygen evolving complex poised in a variety of S 2 state forms giving rise to g = 2 multiline EPR signals. A previous study (Lorigan and Britt (1994) Biochemistry 33: 12072–12076) showed that for PS II membranes treated with 5% ethanol, the S 2 state Mn cluster relaxes via the Orbach spin-lattice relaxation mechanism, where the relaxation is enhanced via phonon scattering off an excited state spin manifold, in this case at an energy of Δ = 36.5 cm−1 above the S = 1/2 ground state giving rise to the multiline EPR signal. Parallel experiments are reported for PS II membranes with 5% methanol, treated with ammonia, and following short and long term dark adaptation. In each case, the temperature dependence of the electron spin-lattice relaxation rate is consistent with Orbach relaxation, and the range of excited state energies is relatively narrow (33.8 cm−1 ≤ Δ ≤ 39.7 cm−1). In addition, short term dark adapted (6 min, ‘active state’) PS II membranes show biphasic recovery traces which indicate that a minority fraction of the oxygen evolving complexes are trapped in a form with greatly slowed spin-lattice relaxation.
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    URL: http://dx.doi.org/10.1023/A:1010604607891
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    Reduction of QA in the dark: Another cause of fluorescence Fo increases by high temperatures in higher plants (2000)
    Springer
    Photosynthesis research 63 (2000), S. 23-34 
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    ISSN: 1573-5079
    Keywords: chlororespiration ; pheophytin a ; photosynthesis ; Photosystem II ; potato ; tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Increases in the chlorophyll fluorescence Fo (dark level fluorescence) during heat treatments were studied in various higher plants. Besides the dissociation of light-harvesting chlorophyll a/b protein complexes from the reaction center complex of PS II and inactivation of PS II, dark reduction of QA via plastoquinone (PQ) seemed to be related to the Fo increase at high temperatures. In potato leaves or green tobacco cultured cells, a part of the Fo increase was quenched by light, reflecting light-induced oxidation of QA - which had been reduced in the dark at high temperatures. Appearance of the Fo increase due to QA reduction depended on the plant species, and the mechanisms for this are proposed. The reductants seemed to be already present and formed by very brief illumination of the leaves at high temperatures. A ndhB-less mutant of tobacco showed that complex I type NAD(P)H dehydrogenase is not involved in the heat-induced reduction of QA. Quite strong inhibition of the QA reduction by diphenyleneiodonium suggests that a flavoenzyme is one of the electron mediator to PQ from the reductant in the stroma. Reversibility of the heat-induced QA reduction suggests that an enzyme(s) involved is activated at high temperatures and mostly returns to an inactive form at room temperature (25 °C).
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    URL: http://dx.doi.org/10.1023/A:1006350706802
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    The IdiA protein of Synechococcus sp. PCC 7942 functions in protecting the acceptor side of Photosystem II under oxidative stress (2000)
    Springer
    Photosynthesis research 63 (2000), S. 145-157 
    Details
    ISSN: 1573-5079
    Keywords: cyanobacteria ; IdiA ; oxidative stress ; Photosystem II ; PsbO ; Synechococcus sp. PCC 7942 and PCC 6301
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from QA, gave a decreased O2 evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage.
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    URL: http://dx.doi.org/10.1023/A:1006322925324
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    Investigation of the plastoquinone pool size and fluorescence quenching in thylakoid membranes and Photosystem II (PS II) membrane fragments (2000)
    Springer
    Photosynthesis research 63 (2000), S. 171-182 
    Details
    ISSN: 1573-5079
    Keywords: Chlorophyll fluorescence ; non-photochemical quenching ; Photosystem II ; thylakoid membranes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The efficiency of oxidized endogenous plastoquinone-9 (PQ-9) as a non-photochemical quencher of chlorophyll fluorescence has been analyzed in spinach thylakoids and PS II membrane fragments isolated by Triton X-100 fractionation of grana stacks. The following results were obtained: (a) After subjection of PS II membrane fragments to ultrasonic treatment in the presence of PQ-9, the area over the induction curve of chlorophyll fluorescence owing to actinic cw light increases linearly with the PQ-9/PS II ratio in the reconstitution assay medium; (b) the difference of the maximum fluorescence levels, Fmax, of the induction curves, measured in the absence and presence of DCMU, is much more pronounced in PS II membrane fragments than in thylakoids; (c) the ratio Fmax(-DCMU)/Fmax(+DCMU) increases linearly with the content of oxidized PQ-9 that is varied in the thylakoids by reoxidation of the pool after preillumination and in PS II membrane fragments by the PQ-9/PS II ratio in the reconstitution assay; (d) the reconstitution procedure leads to tight binding of PQ-9 to PS II membrane fragments, and PQ-9 cannot be replaced by other quinones; (e) the fluorescence quenching by oxidized PQ-9 persists at low temperatures, and (f) oxidized PQ-9 preferentially affects the F695 of the fluorescence emission spectrum at 77 K. Based on the results of this study the oxidized PQ-9 is inferred to act as a non-photochemical quencher via a static mechanism. Possible implications for the nature of the quenching complex are discussed.
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    Inhibition of Photosystem II activity by saturating single turnover flashes in calcium-depleted and active Photosystem II (2000)
    Springer
    Photosynthesis research 63 (2000), S. 209-216 
    Details
    ISSN: 1573-5079
    Keywords: Ca2+-depletion ; charge recombination ; photoinhibition ; Photosystem II ; QA midpoint potential
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Inhibition of Photosystem II (PS II) activity induced by continuous light or by saturating single turnover flashes was investigated in Ca2+-depleted, Mn-depleted and active PS II enriched membrane fragments. While Ca2+- and Mn-depleted PS II were more damaged under continuous illumination, active PS II was more susceptible to flash-induced photoinhibition. The extent of photoinactivation as a function of the duration of the dark interval between the saturating single turnover flashes was investigated. The active centres showed the most photodamage when the time interval between the flashes was long enough (32 s) to allow for charge recombination between the S2 or S3 and QB − to occur. Illumination with groups of consecutive flashes (spacing between the flashes 0.1 s followed by 32 s dark interval) resulted in a binary oscillation of the loss of PS II-activity in active samples as has been shown previously (Keren N, Gong H, Ohad I (1995), J Biol Chem 270: 806–814). Ca2+- and Mn-depleted PS II did not show this effect. The data are explained by assuming that charge recombination in active PS II results in a back reaction that generates P680 triplet and thence singlet oxygen, while in Ca2+- and Mn-depleted PS II charge recombination occurs through a different pathway, that does not involve triplet generation. This correlates with an up-shift of the midpoint potential of QA in samples lacking Ca2+ or Mn that, in term, is predicted to result in the triplet generating pathway becoming thermodynamically less favourable (G.N. Johnson, A.W. Rutherford, A. Krieger, 1995, Biochim. Biophys. Acta 1229, 201–207). The diminished susceptibility to flash-induced photoinhibition in Ca2+- and Mn-depleted PS II is attributed at least in part to this mechanism.
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    Primary charge separation in Photosystem II (2000)
    Springer
    Photosynthesis research 63 (2000), S. 195-208 
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    ISSN: 1573-5079
    Keywords: charge separation ; disorder ; exciton interaction ; Photosystem II ; reaction center
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this Minireview, we discuss a number of issues on the primary photosynthetic reactions of the green plant Photosystem II. We discuss the origin of the 683 and 679 nm absorption bands of the PS II RC complex and suggest that these forms may reflect the single-site spectrum with dominant contributions from the zero-phonon line and a pronounced ∼80 cm−1 phonon side band, respectively. The couplings between the six central RC chlorins are probably very similar and, therefore, a `multimer' model arises in which there is no `special pair' and in which for each realization of the disorder the excitation may be dynamically localized on basically any combination of neighbouring chlorins. The key features of our model for the primary reactions in PS II include ultrafast (〈500 fs) energy transfer processes within the multimer, `slow' (∼20 ps) energy transfer processes from peripheral RC chlorophylls to the RC multimer, ultrafast charge separation (〈500 fs) with a low yield starting from the singlet-excited `accessory' chlorophyll of the active branch, cation transfer from this `accessory' chlorophyll to a `special pair' chlorophyll and/or charge separation starting from this `special pair' chlorophyll (∼8 ps), and slow relaxation (∼50 ps) of the radical pair by conformational changes of the protein. The charge separation in the PS II RC can probably not be described as a simple trap-limited or diffusion-limited process, while for the PS II core and larger complexes the transfer of the excitation energy to the PS II RC may be rate limiting.
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    Hexose transporters of tomato: molecular cloning, expression analysis and functional characterization (2000)
    Springer
    Plant molecular biology 44 (2000), S. 687-697 
    Details
    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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    Functional analysis of the PsbX protein by deletion of the corresponding gene in Synechocystis sp. PCC 6803 (2000)
    Springer
    Plant molecular biology 44 (2000), S. 815-827 
    Details
    ISSN: 1573-5028
    Keywords: 4.1 kDa protein ; low-molecular-mass proteins ; oxygen evolution ; Photosystem II ; PSII ; PsbX
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The psbX gene (sml0002) coding for a 4.1 kDa protein in Photosystem II of plants and cyanobacteria was deleted in both wild type and in a Photosystem I-less mutant of the cyanobacterium Synechocystis sp. PCC 6803. Polymerase chain reaction and sequencing analysis showed that the mutants had completely segregated. Deletion of the PsbX protein does not seem to influence growth rate, electron transport or water oxidation ability. Whereas a high light induction of the psbX mRNA could be observed in wild type, deletion of the gene did not lead to high light sensibility. Light saturation measurements and 77K fluorescence measurements indicated a minor disconnection of the antenna in the deletion mutant. Furthermore, fluorescence induction measurements as well as immuno-staining of the D1 protein showed that the amount of Photosystem II complexes in the mutants was reduced by 30%. Therefore, PsbX does not seem to be necessary for the Photosystem II electron transport, but directly or indirectly involved in the regulation of the amount of functionally active Photosystem II centres in Synechocystis sp. PCC 6803.
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    Substrate specificity of yeast invertase in transglycosylation reactions (2000)
    Springer
    Chemistry of natural compounds 36 (2000), S. 88-89 
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    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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    Identification of a fungal triacetylfusarinine C siderophore transport gene (TAF1) in Saccharomyces cerevisiae as a member of the major facilitator superfamily (1999)
    Springer
    BioMetals 12 (1999), S. 301-306 
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    ISSN: 1572-8773
    Keywords: iron ; siderophores ; transport ; Saccharomyces cerevisiae ; fungi
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Transport proteins of microorganisms may either belong to the ATP-binding cassette (ABC) superfamily or to the major facilitator (MFS)-superfamily. MFS transporters are single-polypeptide membrane transporters that transport small molecules via uniport, symport or antiport mechanisms in response to a chemiosmotic gradient. Although Saccharomyces cerevisiae is a non-siderophore producer, various bacterial and fungal siderophores can be utilized as an iron source. From yeast genome sequencing data six genes of the unknown major facilitator (UMF) family were known of which YEL065w Sce was recently identified as a transporter for the bacterial siderophore ferrioxamine B (Sit1p). The present investigation shows that another UMF gene, YHL047c Sce, encodes a transporter for the fungal siderophore triacetylfusarinine C. The gene YHL047c Sce (designated TAF1) was disrupted using the kanMX disruption module in a fet3 background (strain DEY 1394 Δfet3), possessing a defect in the high affinity ferrous iron transport. Growth promotion assays and transport experiments with 55Fe-labelled triacetylfusarinine C showed a complete loss of iron utilization and uptake in the disrupted strain, indicating that TAF1 is the gene for the fungal triacetylfusarinine transport in Saccharomyces cerevisiae and possibly in other siderophore producing fungi.
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    Interaction of Saccharomyces cerevisiae with gold: toxicity and accumulation (1999)
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    BioMetals 12 (1999), S. 289-294 
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    ISSN: 1572-8773
    Keywords: accumulation ; gold ; proton efflux ; Saccharomyces cerevisiae ; toxicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract This paper examines the effects of ionic gold on Saccharomyces cerevisiae, as determined by long-term (growth in gold-containing media) and short-term interactions (H+ efflux activity). An increasing gold concentration inhibited growth and at 〈0.2 mM Au, growth was not observed. Transmission electron microscopy revealed no differences in ultrastructure but fine electron dense particles were observed in unstained preparations from gold-containing medium. After glucose addition (to 10mM) to starved suspensions of S. cerevisiae, glucose-dependent reduction of external pH occurred as the cells extruded protons. In the presence of increasing gold concentrations, the lag time before proton extrusion did not change but the rate and duration decreased significantly with a marked influence on proton efflux rate being observed at ≤ 10 μM. Extension of preincubation time of yeast cells in gold-containing medium resulted in a decreasing proton efflux rate and colloidal phase formation in the cell suspensions, the time between gold addition and the beginning of colloidal phase formation depending on the gold concentration used. Both Ca and Mg enhanced the inhibitory effect of gold on the yeast cells with Ca showing a stronger inhibitory effect than Mg.
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    Cysteine uptake by Saccharomyces cerevisiae is accomplished by multiple permeases (1999)
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    Current genetics 35 (1999), S. 609-617 
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    ISSN: 1432-0983
    Keywords: Key words Cysteine uptake ; Amino-acid permeases ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Uptake by Saccharomyces cerevisiae of the sulphur-containing amino acid L-cysteine was found to be non-saturable under various conditions, and uptake kinetics suggested the existence of two or more transport systems in addition to the general amino-acid permease, Gap1p. Overexpression studies identified BAP2, BAP3, AGP1 and GNP1 as genes encoding transporters of cysteine. Uptake studies with disruption mutants confirmed this, and identified two additional genes for transporters of cysteine, TAT1 and TAT2, both very homologous to BAP2, BAP3, AGP1 and GNP1. While Gap1p and Agp1p appear to be the main cysteine transporters on the non-repressing nitrogen source proline, Bap2p, Bap3p, Tat1p, Tat2p, Agp1p and Gnp1p are all important for cysteine uptake on ammonium-based medium. Furthermore, whereas Bap2p, Bap3p, Tat1p and Tat2p seem most important under amino acid-rich conditions, Agp1p contributes significantly when only ammonium is present, and Gnp1p only contributes under the latter condition.
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    Starvation in yeast increases non-adaptive mutation (1999)
    Springer
    Current genetics 35 (1999), S. 77-81 
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    ISSN: 1432-0983
    Keywords: Key words Adaptive mutations ; 6-N-hydroxylaminopurine ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The frequency of reversion in a histidine-requiring mutant of Saccharomyces cerevisiae increases about ten-fold in stationary cells during histidine starvation. Histidine starvation enhances a similar frequency of reversion in a tryptophan-requiring mutant. Starvation, therefore, enhances mutation frequencies in a non-adaptive manner. The base analogue 6-N-hydroxylaminopurine (HAP) added prior to plating on medium with limited histidine strongly increases reversion of the histidine mutant. HAP-induced reversion increases further in stationary starving cells with the same kinetics as that which increases spontaneous reversion. Adding HAP to the stationary starving cells does not produce any effect.
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    Strand interruptions confer strand preference during intracellular correction of a plasmid-borne mismatch in Saccharomyces cerevisiae (1999)
    Springer
    Current genetics 35 (1999), S. 499-505 
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    ISSN: 1432-0983
    Keywords: Key words Heteroduplex repair ; Strand discrimina-tion ; Strand interruptions ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Site-directed mutagenesis was used to construct yeast centromere plasmids in which a strand nick or gap could be placed 5′ or 3′, on either strand, to a reporter gene (SUP4-o) carrying defined base mismatches. The plasmids were then transformed into yeast cells and the direction and efficiency of mismatch repair were assayed by scoring colouring of the transformant colonies. Strands that were nicked were consistently corrected more often than intact strands, but the effect was very small. However, placement of a small gap at the same positions as the nicks resulted in a marked increase in selection for the gapped strand and an enhanced efficiency of mismatch repair. Both the preference for the gapped strand and correction of the mismatch were offset by deletion of the mismatch repair gene PMS1. Together, the results suggest that strand interruptions can direct intracellular mismatch correction of plasmid-borne base mispairs in yeast.
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    Analysis of the genes activated by the FLO8 gene in Saccharomyces cerevisiae (1999)
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    Current genetics 36 (1999), S. 256-261 
    Details
    ISSN: 1432-0983
    Keywords: Key wordsFLO8 ; Transcriptional regulation ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract It is thought that the FLO8 gene encodes a transcriptional activator of the dominant flocculation gene FLO1 in Saccharomycescerevisiae. To determine other genes which are regulated by FLO8, a detailed comparison of the transcripts from the FLO8 and Δflo8 strains was carried out. In addition to the FLO1 gene, it was found that transcription of the FLO11 and STA1 genes is positively regulated by FLO8. In flo8 strains, not only transcripts of the FLO11, STA1, and FLO1 genes but also invasive growth, extracellular glucoamylase production, and flocculation were undetected. From these results, it is suggested that FLO8 regulates these characteristics via the transcriptional regulation of the FLO11, STA1, and FLO1 genes.
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    Mutant allele pso7-1, that sensitizes Saccharomyces cerevisiae to photoactivated psoralen, is allelic with COX11, encoding a protein indispensable for a functional cytochrome c oxidase (1999)
    Springer
    Current genetics 36 (1999), S. 124-129 
    Details
    ISSN: 1432-0983
    Keywords: Key words Psoralen sensitivity ; Cytochrome oxidase ; Saccharomyces cerevisiae ; Oxidative stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast gene PSO7 was cloned from a genomic library by complementation of the pso7-1 mutant's sensitivity phenotype to 4-nitroquinoline-1-oxide (4NQO). Sequence analysis revealed that PSO7 is allelic to the 1.1-kb ORF of the yeast gene COX11 which is located on chromosome XVI and encodes a protein of 28-kDa localized in the inner mitochondrial membrane. Allelism of PSO7/COX11 was verified by non-complementation of 4NQO-sensitivity in diploids homo- and hetero-allelic for the pso7-1 and cox11::TRP1 mutant alleles. Sensitivity to 4NQO was the same in exponentially growing cells of the pso7-1 mutant and the cox11::TRP1 disruptant. Allelism of COX11 and PSO7 indicates that the pso7 mutant's sensitivity to photoactivated 3-carbethoxypsoralen and to 4NQO is not caused by defective DNA repair, but rather is due to an altered metabolism of the pro-mutagen 4NQO in the absence of cytochrome oxidase (Cox) in pso7-1/cox11::TRP1 mutants/disruptants. Lack of Cox might also lead to a higher reactivity of the active oxygen species produced by photoactivated 3-carbethoxypsoralen. The metabolic state of the cells is important for their sensitivity phenotype since the largest enhancement of sensitivity to 4NQO between wild-type (WT) and the pso7 mutant occurs in exponentially growing cells, while cells in stationary phase or growing cells in phosphate buffer have the same 4NQO resistance, irrespective of their WT/mutant status. Strains containing the pso7-1 or cox11::TRP1 mutant allele were also sensitive to the oxidative stress-generating agents H2O2 and paraquat. Mutant pso7-1, as well as disruptant cox11::TRP1, harboured mitochondria that in comparison to WT contained less than 5% and no detectable Cox activity, respectively.
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    Comparative effects of Saccharomyces cerevisiae cultivation under copper stress on the activity and kinetic parameters of plasma-membrane-bound H+-ATPases PMA1 and PMA2 (1999)
    Springer
    Archives of microbiology 171 (1999), S. 273-278 
    Details
    ISSN: 1432-072X
    Keywords: Key words Plasma membrane H+-ATPase ; PMA1 ; ATPase ; PMA2 ATPase ; Saccharomyces cerevisiae ; Copper stress ; Copper tolerance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The major yeast plasma membrane H+-ATPase is encoded by the essential PMA 1 gene. The PMA 2 gene encodes an H+-ATPase that is functionally interchangeable with the one encoded by PMA 1 , but it is expressed at a much lower level than the PMA 1 gene and it is not essential. Using genetically manipulated strains of Saccharomyces cerevisiae that exclusively synthesize PMA1 ATPase or PMA2 ATPase under control of the PMA1 promoter, we found that yeast cultivation under mild copper stress leads to a similar activation of PMA2 and PMA1 isoforms. At high inhibitory copper concentrations (close to the maximum that allowed growth), ATPase activity was reduced from maximal levels; this decrease in activity was less important for PMA2 ATPase than for PMA1 ATPase. The higher tolerance to high copper stress of the artificial strain synthesizing PMA2 ATPase exclusively, as compared to that synthesizing solely PMA1 ATPase, correlated both with the lower sensitivity of PMA2 ATPase to the deleterious effects of copper in vivo and with its higher apparent affinity for MgATP, and suggests that plasma membrane H+-ATPase activity plays a role in yeast tolerance to copper.
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    A strange calmodulin of yeast (1999)
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    Molecular and cellular biochemistry 190 (1999), S. 47-54 
    Details
    ISSN: 1573-4919
    Keywords: calmodulin ; yeast calmodulin ; Ca2+ binding ; Ca2+ binding protein ; Saccharomyces cerevisiae ; interdomain interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Calmodulin of Saccharomyces cerevisiae has different Ca2+ binding properties from other calmodulins. We previously reported that the maximum number of Ca2+ binding was 3 mol/mol and the fourth binding site was defective, which was different from 4 mol/mol for others. Their macroscopic dissociation constants suggested the cooperative three Ca2+ bindings rather than a pair of cooperative two Ca2+ bindings of ordinary calmodulin. Here we present evidence for yeast calmodulin showing the intramolecular close interaction between the N-terminal half domain and the C-terminal half domain, while the two domains of ordinary calmodulin are independent of each other. We will discuss the relationship of the shape and the shape change caused by the Ca2+ binding to the enzyme activation in yeast. The functional feature of calmodulin in yeast will also be considered, which might be different from the one of vertebrate calmodulin.
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    Characterization of Saccharomyces cerevisiae strains expressing ira1 mutant alleles modeled after disease-causing mutations in NF1 (1999)
    Springer
    Molecular and cellular biochemistry 202 (1999), S. 109-118 
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    ISSN: 1573-4919
    Keywords: NF1 mutations ; IRA1 ; Saccharomyces cerevisiae ; RAS2 ; GAP activity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The 2818 amino acids of neurofibromin, the product of the human NF1 gene, include a 230 amino acid Ras-GAP related domain (GRD). Functions which may be associated with the rest of the protein remain unknown. However, many NF1 mutations in neurofibromatosis 1 patients are found downstream of the GRD, suggesting that the C-terminal region of the protein is also functionally important. Since the C-terminal region of neurofibromin encompassing these mutations is homologous with the corresponding regions in the two Saccharomyces cerevisiae Ras-GAPs, Ira1p and Ira2p, we chose yeast as a model system for functional exploration of this region (Ira-C region). Three missense mutations that affect the Ira-C region of NF1 were used as a model for the mutagenesis of IRA1. The yeast phenotypes of heat shock sensitivity, iodine staining, sporulation efficiency, pseudohyphae formation, and GAP activity were scored. Even though none of the mutations directly affected the Ira1p-GRD, mutations at two of the three sites resulted in a decrease in the GAP activity present in ira1 cells. The third mutation appeared to disassociate the phenotypes of sporulation ability and GAP activity. This and other evidence suggest an effector function for Ira1p.
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    Direct observation of oxidative stress on the cell wall of Saccharomyces cerevisiae strains with atomic force microscopy (1999)
    Springer
    Molecular and cellular biochemistry 201 (1999), S. 17-24 
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    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; atomic force microscope ; bioscope ; organic synthesis ; molecular biology ; oxidative stress ; pore enlargement ; cell wall ; baker's yeast ; biotechnology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We imaged pores on the surface of the cell wall of three different industrial strains of Saccharomyces cerevisiae using atomic force microscopy. The pores could be enlarged using 10 mM diamide, an SH residue oxidant that attacks surface proteins. We found that two strains showed signs of oxidative damage via changes in density and diameter of the surface pores. We found that the German strain was resistant to diamide induced oxidative damage, even when the concentration of the oxidant was increased to 50 mM. The normal pore size found on the cell walls of American strains had diameters of about 200nm. Under conditions of oxidative stress the diameters changed to 400nm. This method may prove to be a useful rapid screening process (45-60 min) to determine which strains are oxidative resistant, as well as being able to screen for groups of yeast that are sensitive to oxidative stress. This rapid screening tool may have direct applications in molecular biology (transference of the genes to inside of living cells) and biotechnology (biotransformations reactions to produce chiral synthons in organic chemistry.
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    Characterization of the Oxaloacetate Decarboxylase and Pyruvate Kinase-like Activities of Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinases (1999)
    Springer
    The protein journal 18 (1999), S. 659-664 
    Details
    ISSN: 1573-4943
    Keywords: Phosphoenolpyruvate carboxykinase ; oxaloacetate decarboxylase ; pyruvate kinase-like activity ; Anaerobiospirillum succiniciproducens ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Two members of the ATP-dependent class of phosphoenolpyruvate carboxykinases (PEPCKs) (Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens) have been comparatively studied with regard to their oxaloacetate (OAA) decarboxylase and pyruvate kinase-like activities. The pyruvate kinase-like activities were dependent on the presence of Mn2+; at the same concentrations Mg2+ was not effective. These activities were synergistically activated by a combination of both metal ions. V max for these activities in A. succiniciproducens and S. cerevisiae PEPCKs was 0.13% and 1.2% that of the principal reaction, respectively. The OAA decarboxylase activity was nucleotide independent and, with decreasing order of effectiveness, these activities were supported by Mn2+ and Mg2+. AMP is an activator of these reactions. V max for the OAA decarboxylase activities in A. succiniciproducens and S. cerevisiae PEPCKs was 4% and 0.2% that of the PEP-forming reaction, respectively.
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    Genetic evidence for interaction between components of the yeast 26S proteasome: combination of a mutation in RPN12 (a lid component gene) with mutations in RPT1 (an ATPase gene) causes synthetic lethality (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 145-153 
    Details
    ISSN: 1617-4623
    Keywords: Key words Proteasome ; Synthetic lethality ; Saccharomyces cerevisiae ; AAA-ATPase ; 19S Regulatory particle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The 19S regulatory particle of the yeast 26S proteasome consists of six related ATPases (Rpt proteins) and at least 11 non-ATPase proteins (Rpn proteins). RPN12 (formerly NIN1) encodes an Rpn component of the 19S regulatory particle and is essential for growth. To determine which subunit(s) of the 26S proteasome interact(s) with Rpn12, we attempted to screen for mutations that cause synthetic lethality in the presence of the rpn12-1 (formerly nin1-1) mutation. Among the candidates recovered was a new allele of RPT1 (formerly CIM5). This mutant allele was designated rpt1-2; on its own this mutation caused no phenotypic change, whereas the rpn12-1 rpt1-2 double mutant was lethal, suggesting a strong interaction between Rpn12 and Rpt1. The site of the rpt1-2 mutation was determined by DNA sequencing of the RPT1 locus retrieved from the mutant, and a single nucleotide alteration was found. This changes amino acid 446 of the RPT1 product from alanine to valine. The alanine residue is conserved in all Rpt proteins, except Rpt5, but no function has yet been assigned to the region that contains it. We propose that this region is necessary for Rpt1 to interact with Rpn12. The terminal phenotype of the rpn12-1 rpt1-2 double mutant was not cell cycle specific, suggesting that in the double mutant cells the function of the 26S proteasome is completely eliminated, thereby inducing multiple defects in cellular functions.
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    URL: http://dx.doi.org/10.1007/s004380051069
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    Cat8p, the activator of gluconeogenic genes in Saccharomyces cerevisiae, regulates carbon source-dependent expression of NADP-dependent cytosolic isocitrate dehydrogenase (Idp2p) and lactate permease (Jen1p) (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 869-875 
    Details
    ISSN: 1617-4623
    Keywords: Key wordsCAT8 ; Transcriptional regulation ; IDP2 ; JEN1 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast transcriptional activator Cat8p has been identified as a factor that is essential for the derepression of genes involved in gluconeogenesis (like FBP1, PCK1, ACR1, ICL1 and MLS1) when only non-fermentable carbon sources are provided. Cat8p-dependent expression is mediated by cis-acting elements in the respective promoters, which are named UAS/CSREs (upstream activating sequence/carbon source responsive element). To establish whether the function of Cat8p is restricted to the activation of gluconeogenesis or is also involved in the regulation of a greater variety of genes, we investigated the transcriptional regulation of two genes, IDP2 and JEN1, which exhibit a similar expression pattern to gluconeogenic genes, although IDP2 at least is not linked directly to the gluconeogenic pathway. We identified functional UAS/CSRE elements in the promoters of both genes. Expression studies revealed that JEN1 is regulated negatively by the repressors Mig1p and Mig2p, and that Cat8p is needed for full derepression of the gene under non-fermentative growth conditions. Furthermore, we showed that Mig2p is also involved in the repression of CAT8 itself. The results presented in this study support a model in which Cat8p-dependent gene activation is not restricted to gluconeogenesis, but targets a wide variety of genes which are strongly derepressed under non-fermentative growth conditions.
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    URL: http://dx.doi.org/10.1007/s004380051152
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    Yeast genes GIS1–4: multicopy suppressors of the Gal− phenotype of snf1 mig1 srb8/10/11 cells (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 589-599 
    Details
    ISSN: 1617-4623
    Keywords: Key words Ras/cAMP pathway ; Saccharomyces cerevisiae ; Snf1 ; Mig1 ; Mediator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cyclin C and the cyclin C-dependent protein kinase are associated with the RNA polymerase II Mediator complex, which regulates initiation of transcription in response to signals from activators and repressors bound to upstream promoter elements. Disruption of the corresponding genes, SRB11 and SRB10, in budding yeast causes a reduction in expression of the GAL genes, which is particularly pronounced in a mig1 snf1 background. We have screened two yeast genomic libraries for genes that can suppress this phenotype when overexpressed. Seven suppressor genes were identified, GIS1–7. GIS1 encodes one of two related zinc-finger proteins, which also share two other highly conserved domains present in several eukaryotic transcription factors. GIS2 encodes a homologue of the mammalian CNBP and fission yeast Byr3 proteins. GIS3 and GIS4 predict proteins with no obvious similarities to any known proteins. GIS5–7 are identical to the previously described genes PDE2, SGE1 and TUB3, respectively. None of the suppressor genes seem to be involved in Mediator function. Instead, we find that the GIS1, GIS2 and GIS4 genes interact with the CDC25 gene, indicating a possible involvement of these genes in the RAS/cAMP signaling pathway.
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    URL: http://dx.doi.org/10.1007/s004380051121
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    Genetic evidence for interactions between yeast importin α (Srp1p) and its nuclear export receptor, Cse1p (1999)
    Springer
    Molecular genetics and genomics 261 (1999), S. 788-795 
    Details
    ISSN: 1617-4623
    Keywords: Key words Cse1p ; Srp1p ; Importin ; Nuclear transport ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast Srp1p protein functions as an import receptor for proteins bearing basic nuclear localization signals. Cse1p, the yeast homolog of mammalian CAS, recycles Srp1p back to the cytoplasm after import substrates have been released into the nucleoplasm. In this report we describe genetic interactions between SRP1 and CSE1. Results from genetic suppression and synthetic lethality studies demonstrate that these gene products interact to ensure accurate chromosome segregation. We also describe new mutant alleles of CSE1 and analyze a new temperature-sensitive allele of CSE1, cse1-2. This allele causes high levels of chromosome missegregation and cell cycle arrest during mitosis at the nonpermissive temperature.
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    URL: http://dx.doi.org/10.1007/s004380050022
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    The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 332-341 
    Details
    ISSN: 1617-4623
    Keywords: Key words Leucine transport ; Saccharomyces cerevisiae ; Trifluoroleucine resistance ; LEP1 ; SAC3
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine. A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize l-leucine as sole nitrogen source and very low levels of [14C]l-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [14C]l-leucine uptake. Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine. The complementing gene, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation. The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin mutant containing the allele act1-1. Sac3p has been previously shown to be localized in the nucleus. When a lep1 mutant was crossed with a sac3 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic.
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    URL: http://dx.doi.org/10.1007/s004380051091
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    Galactose induction in yeast involves association of Gal80p with Gal1p or Gal3p (1999)
    Springer
    Molecular genetics and genomics 261 (1999), S. 495-507 
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    ISSN: 1617-4623
    Keywords: Key wordsKluyveromyces lactis ; Saccharomyces cerevisiae ; GAL1 ; GAL80 ; Protein interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interacting with Gal80p – a function that can also served by Gal3p – and it catalyzes the formation of galactose-1-phosphate. Recently, we and others have presented biochemical evidence for complex formation between Gal1p and Gal80p. Here, we extend these data and present genetic evidence for an interaction between Gal1p and Gal80p in vivo, using a two-hybrid assay. Interaction between Gal1p and Gal80p depends on the presence of galactose, but not on the catalytic activity of Gal1p. A new class of Kluyveromyces lactis mutants was isolated, designated Klgal1-m, which have lost the derepressing activity but retain galactokinase activity, indicating that the two Gal1p activities are functionally independent. The KlGal1-m proteins are defective in their ability to interact with Gal80p in a two-hybrid assay. The locations of gal1-m mutations identify putative interaction sites in Gal1p and Gal80p. A dominant mutation, KlGAL1-d, leads to a high level of constitutive expression of genes of the galactose pathway. The behavior of chimeric proteins consisting of Gal3p and KlGal1p sequences indicates that both the N-terminal and C-terminal halves of KlGal1p are involved in specific interaction with KlGal80p.
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    URL: http://dx.doi.org/10.1007/s004380050993
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    The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7) (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 437-447 
    Details
    ISSN: 1617-4623
    Keywords: Key words Oxidative stress signalling ; Mitochondria ; Pos9 (Skn7) ; Ccp1 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation group fap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1 W191F , which is characterized by a 104-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.
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    URL: http://dx.doi.org/10.1007/s004380051103
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    A human gene, hSGT1, can substitute for GCR2, which encodes a general regulatory factor of glycolytic gene expression in Saccharomyces cerevisiae (1999)
    Springer
    Molecular genetics and genomics 260 (1999), S. 535-540 
    Details
    ISSN: 1617-4623
    Keywords: Key words Gene expression ; Glycolysis ; GCR ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To determine whether similar regulatory mechanisms control the expression of glycolytic genes in yeast and human cells, we screened a human brain cDNA library for clones which complement the growth defect of the gcr2 mutant of Saccharomyces cerevisiae, and isolated hSGT1 (human suppressor of GCR two). Further work confirmed that the rescue of growth was associated with recovery of glycolytic enzyme activities, and that hSGT1 did not complement the growth defect of a gcr1 mutant. A hybrid protein comprising hSgt1p and the DNA-binding domain of Gal4p (GBD) activated a GAL1-lacZ reporter gene fusion, suggesting that the cloned gene may be a transcriptional activator. Two-hybrid experiments in yeast also indicate that hSgt1p interacts with Gcr1p. Northern analysis showed that hSGT1 is highly expressed in muscle and heart. Although the predicted amino acid sequence of hSgt1p does not display significant similarity to Gcr2p, we speculate that their functions may be analogous.
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    URL: http://dx.doi.org/10.1007/s004380050926
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    The Saccharomyces cerevisiae RAD54 gene is important but not essential for natural homothallic mating-type switching (1999)
    Springer
    Molecular genetics and genomics 260 (1999), S. 551-558 
    Details
    ISSN: 1617-4623
    Keywords: Key wordsRAD54 ; Saccharomyces cerevisiae ; Recombination ; Mating-type ; DNA repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Homothallic Saccharomyces cerevisiae strains switch their mating-type in a specific gene conversion event induced by a DNA double strand break made by the HO endonuclease. The RAD52 group genes control recombinational repair of DNA double strand breaks, and we examined their role in native homothallic mating-type switching. Surprisingly, we found that the Rad54 protein was important but not essential for mating-type switching under natural conditions. As an upper limit, we estimate that 29% of the rad54 spore clones can successfully switch their mating-type. The RAD55 and RAD57 gene products were even less important, but their presence increased the efficiency of the process. In contrast, the RAD51 and RAD52 genes are essential for homothallic mating-type switching. We propose that mating-type switching in RAD54 mutants occurs stochastically with a low probability, possibly reflecting different states of chromosomal structure.
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    URL: http://dx.doi.org/10.1007/s004380050928
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    Random mutagenesis in the large extrinsic loop E and transmembrane α-helix VI of the CP 47 protein of Photosystem II (1999)
    Springer
    Plant molecular biology 39 (1999), S. 381-386 
    Details
    ISSN: 1573-5028
    Keywords: Photosystem II ; CP 47 ; random mutagenesis ; XL-1 Red ; mutator strains
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The intrinsic chlorophyll-protein CP 47 is a component of Photosystem II which functions in both light-harvesting and oxygen evolution. Using the Escherichia coli mutator strain XL-1 Red, we introduced mutations at 14 sites in the large extrinsic loop E of CP 47 and its adjacent transmembrane α-helix VI. Four mutant cell lines were recovered in which the histidyl residues 455H, 466H and 469H were altered. The cell lines H455T, H455Y, H469Y, and the double mutant F432L,H466R exhibited phenotypes that supported the identification of the histidyl residues 455H, 466H and 469H as chlorophyll ligands. Four additional mutant cell lines were recovered which contained mutations at positions 448R in the large extrinsic loop of CP 47. These mutants, R448K, R448Q, R448S, and R448W, exhibited variable phenotypes ranging from moderate alteration of photoautotrophic growth and oxygen evolution rates to a complete inhibition of these parameters. Those mutants exhibiting photoautotrophic growth and oxygen evolution capability under standard conditions were unable to grow photoautotrophically or evolve oxygen when grown at low chloride concentrations. Finally, a mutant cell line exhibiting a substitution at position 342G was recovered. The mutant G342D exhibited moderate alterations of photoautotrophic growth and oxygen evolution. In addition to these alterations, mutants were recovered in which deletions and insertions (leading to frame shifts) and stop codons were introduced. These mutants uniformly lacked the ability to either grow photoautotrophically or evolve oxygen.
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    Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase (1999)
    Springer
    Plant molecular biology 39 (1999), S. 953-967 
    Details
    ISSN: 1573-5028
    Keywords: Arabidopsis thaliana ; heterologous expression ; isoprenoids ; mevalonate diphosphate decarboxylase ; sterols ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.
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    The effect of copper on chlorophyll organization during greening of barley leaves (1999)
    Springer
    Photosynthesis research 62 (1999), S. 165-174 
    Details
    ISSN: 1573-5079
    Keywords: chlorophyll fluorescence ; oxygen evolution ; photoacoustic ; photodamage ; Photosystem II
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of copper on chlorophyll organization and function during greening of barley was examined, using chlorophyll fluorescence and photoacoustic techniques. Copper was found to inhibit pigment accumulation and to retard chlorophyll integration into the photosystems, as evident from low temperature (77 K) fluorescence spectra. Resolution of the minimal fluorescence (F0) into active and inactive parts, indicated a higher inactive fraction with copper treatment. This was attributed to chlorophyll molecules which failed to integrate normally, a conclusion supported by the longer fluorescence lifetime observed in copper treated plants. A lower ratio of chlorophyll a to b and fluorescence induction transients, showing accelerated Photosystem II closure, both indicate that copper treatment resulted in a larger light-harvesting antenna. Another effect of copper treatment was the suppression of oxygen evolution, indicating a decrease in photosynthetic capacity. We suggest that the non-integrated chlorophyll fraction sensitizes photodamage in the membrane, contributing to disruption of electron flow and pigment accumulation.
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    Dark reoxidation of the plastoquinone-pool is mediated by the low-potential form of cytochrome b-559 in spinach thylakoids (1999)
    Springer
    Photosynthesis research 62 (1999), S. 273-279 
    Details
    ISSN: 1573-5079
    Keywords: plastoquinone ; cytochrome b-559 ; thylakoid membrane ; Photosystem II ; chlororespiration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have found that in isolated spinach thylakoids, plastoquinone-pool (PQ-pool), after its photoreduction, undergoes dark-reoxidation with the half-time of τ1/2 = 43 ± 3 s. To explain the observed rates of PQ-pool reoxidation, a nonenzymatic plastoquinol (PQH2) autoxidation under molecular oxygen and an enzymatic oxidation by the low-potential form of cytochrome b-559 (cyt. b-559LP), as the postulated PQ-oxidase in chlororespiration, were investigated. It was found that the autoxidation rate of PQH2 in organic solvents and liposomes was too low to account for the observed oxidation rate of PQH2 in thylakoids. The rate of cyt. b-559LP autoxidation in isolated Photosystem II was found to be similar (τ1/2 = 26 ± 5 s) to that of the PQ-pool. This suggests that the LP form of cyt. b-559 is probably responsible for the PQ-oxidase activity observed during chlororespiration.
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    Effects of anaerobiosis as probed by the polyphasic chlorophyll a fluorescence rise kinetic in pea (Pisum sativum L.) (1999)
    Springer
    Photosynthesis research 62 (1999), S. 67-83 
    Details
    ISSN: 1573-5079
    Keywords: anaerobiosis ; chlorophyll fluorescence ; chlororespiration ; fluorescence induction ; Photosystem II ; Pisum sativum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We analysed the changes of the chlorophyll (Chl) a fluorescence rise kinetic (from 50 μs to 1 s) that occur when leaves or chloroplasts of pea ( Pisum sativum L.) are incubated under anaerobic conditions in the dark. In control leaves, Chl a fluorescence followed a typical O-J-I-P polyphasic rise [Strasser et al. (1995) Photochem Photobiol 61: 32–42]. Anaerobiosis modified the shape of the transient with the main effect being a time-dependent increase in the fluorescence yield at the J-step (2 ms). Upon prolongation of the anaerobic treatment (〉 60 min), the O-J-I-P fluorescence rise was eventually transformed to an O-J (J = P) rise. A similar transformation was observed when pea leaves were treated with DCMU or sodium dithionite. Anaerobiosis resulted in a 10–20% reduction in the maximum quantum yield of the primary photochemistry of Photosystem II, as measured by the ratio of the maximal values of variable and total fluorescence (FV/FM). When the leaves were returned to the air in the dark, the shape of the fluorescence transient showed a time-dependent recovery from the anaerobiosis-induced change. The original O-J-I-P shape could also be restored by illuminating the anaerobically treated samples with far-red light but not with blue or white light. Osmotically broken chloroplasts displayed under anaerobic conditions fluorescence transients similar to those observed in anaerobically treated leaves, but only when they were incubated in a medium comprising reduced pyridine nucleotides (NADPH or NADH). As in intact leaves, illumination of the anaerobically treated chloroplasts by far-red light restored the original O-J-I-P transient, although only in the presence of methyl viologen. The results provide additional evidence for the existence of a chlororespiratory pathway in higher plant cells. Furthermore, they suggest that the J-level of the fluorescence transient is strongly determined by the redox state of the electron carriers at the PS II acceptor side.
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    The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase (1999)
    Springer
    Journal of bioenergetics and biomembranes 31 (1999), S. 95-104 
    Details
    ISSN: 1573-6881
    Keywords: F1-ATPase ; β-barrel domain ; mitochondria ; assembly ; yeast ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The crystal structure of mitochondrial F1-ATPase indicatesthat the α and β subunits fold into a structure defined by threedomains: the top β-barrel domain, the middle nucleotide-binding domain,and the C-terminal α-helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The β-barrel domains of theα and β subunits form a crown structure at the top ofF1, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the β-barrel domain in the α andβ subunits of the yeast Saccharomyces cerevisiae F1,with regard to its folding and assembly, was investigated. The β-barreldomains of yeast F1 α and β subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the β-barrel domain of the β subunit formed a largeaggregate structure, while the domain of the α subunit waspredominately a monomer or dimer. However, coexpression of the β-barreldomain of α subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the β-barrel domain of theα and β subunits interact specifically with each other and thatthese interactions prevent the aggregation of the β-barrel domain of theβ subunit. These results mimic in vivo results and suggest thatthe interactions of the β-barrel domains may be critical during thefolding and assembly of F1.
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    Saké brewing characteristics and multidrug resistance of trichothecin-resistant yeast mutants (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 629-630 
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    ISSN: 1573-0972
    Keywords: Ethanol ; multi-drug resistance ; Saccharomyces cerevisiae ; trichothecin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Trichothecin-resistant mutants were isolated from saké yeast. These mutants were subjected to saké brewing, and showed a higher ethanol productivity than did the parents. They showed multidrug resistance, and resistance to organic compounds. We considered that the higher ethanol productivity of the mutants was related to their resistance to organic compounds and to their ethanol tolerance.
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    The wheat LEA protein Em functions as an osmoprotective molecule in Saccharomyces cerevisiae (1999)
    Springer
    Plant molecular biology 39 (1999), S. 117-128 
    Details
    ISSN: 1573-5028
    Keywords: LEA protein ; osmotic stress ; Saccharomyces cerevisiae ; drought ; salt
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The biased amino acid composition and aperiodic (random coil) configuration of Group 1 late embryogenesis-abundant (LEA) proteins imply that these proteins are capable of binding large amounts of water. While Group 1 LEAs have been predicted to contribute to osmotic stress protection in both embryonic and vegetative tissues, biochemical support has been lacking. We have used Saccharomyces cerevisiae as a model system to test the putative osmoprotective function of a wheat Group 1 LEA protein, Em. We demonstrate that expression of Em protein in yeast cells is not deleterious to growth in media of normal osmolarity and attenuates the growth inhibition normally observed in media of high osmolarity. Enhanced growth is observed in the presence of a variety of osmotically active compounds indicating that Em protein is capable of mitigating the detrimental effect of low water potential in a relatively non-specific manner. These results are the first biochemical demonstration of an osmoprotective function for a Group 1 LEA and suggest that the yeast expression system will be useful in dissecting the mechanism of protection through structure-function studies.
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    Isolation and characterization of chloroplast Photosystem II antenna of spinach by reversed-phase liquid chromatography (1999)
    Springer
    Photosynthesis research 61 (1999), S. 281-290 
    Details
    ISSN: 1573-5079
    Keywords: antenna system ; chlorophyll–proteins ; HPLC ; LHC II ; Photosystem II ; spinach
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The protein components of the Photosystem II antenna system, isolated from spinach thylakoids, have been resolved by reversed-phase high performance liquid chromatography (RP-HPLC) using a butyl-silica stationary phase packed either into analytical or semi-preparative columns. Peak identification has been accomplished by a combination of various SDS–PAGE systems employing either Comassie (or silver) staining or immunological detection using polyclonal antibodies raised against LHC II and against CP29, CP26 and CP24 proteins and by aminoacid microsequence. Moreover, peak identification is consistent with the molecular masses determined by Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS). The developed RP-HPLC method allows the resolution of all the protein components of the Photosystem II major Light Harvesting Complex (LHC II) and minor PS II antenna complex (CP24, CP26 and CP29) from grana membranes (BBY) and estimation of their relative stoichiometry in natural and stressed conditions, avoiding the expensive and time consuming separation procedure by sucrose-gradient ultracentrifugation and isoelectrofocusing.
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    URL: http://dx.doi.org/10.1023/A:1006397419473
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    Hg2+, Cu2+, and Pb2+ -induced changes in Photosystem II photochemical yield and energy storage in isolated thylakoid membranes: A study using simultaneous fluorescence and photoacoustic measurements (1999)
    Springer
    Photosynthesis research 59 (1999), S. 167-174 
    Details
    ISSN: 1573-5079
    Keywords: cyclic electron transport ; fluorescence ; metal ions ; Photosystem II ; thermal dissipation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Simultaneous fluorescence and photoacoustic measurements have been used to study the effects of metal ions (copper, lead, and mercury) during dark incubation of thylakoid membranes. The values of the chlorophyll fluorescence parameters Fo (initial fluorescence yield with the reaction centers in the open state), Fm (maximal fluorescence yield), Ft (steady state fluorescence yield) and the calculated parameters, Φo (maximal quantum yield of Photosystem II photochemistry) and Φt (actual quantum yield of Photosystem II photochemistry), strongly decreased in the presence of the metal ions coinciding with an increase in the non-photochemical deexcitation rate constant k(N). It was observed that photosynthetic energy storage measured by photoacoustic spectroscopy also decreased but a large portion of energy storage remained unaffected even at the highest metal ion concentrations used. A maximal inhibition of photosyntheti c energy storage of 80% and 50% was obtained with Hg2+ and Cu2+-treated thylakoids, respectively, while energy storage was insensitive to Pb2+. The results are consistent with the known predominant inhibition of the donor side of Photosystem II by the metal ions. The insensitive portion of energy storage is attributed to the possible recurrence of cyclic electron transport around Photosystem II that would depend on the extent of inhibition produced on the acceptor side by the metal ion used.
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    URL: http://dx.doi.org/10.1023/A:1006194621553
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    Structural predictions on the 33 kDa extrinsic protein associated to the oxygen evolving complex of photosynthetic organisms (1999)
    Springer
    Photosynthesis research 61 (1999), S. 11-21 
    Details
    ISSN: 1573-5079
    Keywords: manganese-stabilizing protein ; oxygen evolution ; Photosystem II ; protein structure prediction ; PsbO protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Modern computational methods for protein structure prediction have been used to study the structure of the 33 kDa extrinsic membrane protein, associated to the oxygen evolving complex of photosynthetic organisms. A multiple alignment of 14 sequences of this protein from cyanobacteria, algae and plants is presented. The alignment allows the identification of fully conserved residues and the recognition of one deletion and one insertion present in the plant sequences but not in cyanobacteria. A tree of similarity, deduced from pair-wise comparison and cluster analysis of the sequences, is also presented. The alignment and the consensus sequence derived are used for prediction the secondary structure of the protein. This prediction indicates that it is a mainly-beta protein (25–38% of β-strands) with no more than 4% of α-helix. Fold recognition by threading is applied to obtain a topological 2D model of the protein. In this model the secondary structure elements are located, including several highly conserved loops. Some of these conserved loops are suggested to be important for the binding of the 33 kDa protein to Photosystem II and for the stability of the manganese cluster. These structural predictions are in good agreement with experimental data reported by several authors.
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    URL: http://dx.doi.org/10.1023/A:1006265816104
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    Correlations between chlorophyll fluorescence quenching parameters and photosynthesis in a segregating Lycopersicon esculentum × L. peruvianum population as measured under constant conditions (1999)
    Springer
    Photosynthesis research 61 (1999), S. 145-156 
    Details
    ISSN: 1573-5079
    Keywords: chilling ; Photosystem II ; quantum efficiency
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chl fluorescence of mature leaves in low-temperature treated plants was studied under identical measuring conditions in a segregating population of the F3 offspring of a cross between a chilling-tolerant and a chilling-sensitive tomato species. Through recombination of genes involved in photosynthesis, the population revealed a wide, continuous variability of photosynthetic capacity from plants performing much worse to those performing better than the parental lines of the cross. In the parental species, a nearly linear correlation was observed between photochemical chl fluorescence quenching (qP) and O2 evolution over a wide temperature range. Across the F3 generation, still a weak correlation between the two parameters was found at 20 °C, but not at 10 °C, when measured under identical conditions. This indicates that the fraction of open reaction centres could at least in part be adjusted to the photosynthetic capacity of the individual genotype. However, the correlation was so weak, that the previously suggested use of qP as a selection criterion for chilling tolerance of photosynthesis in breeding programs is regarded as doubtful, as long as photosynthesis rates are not measured in addition. Quantum efficiency of Photosystem II (ΦPSII) was strongly dependent on qP both at 20 and at 10 °C measuring temperature and depended on the quantum efficiency of open reaction centres (F′v/F′m) at 20, but not at 10 °C. F′v/F′m, in turn, correlated negatively with the processes of energy dissipation by the mechanisms of non-photochemical quenching (qN), i.e. its fast-relaxing component (qF) and photoinhibitory quenching (qI).
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    URL: http://dx.doi.org/10.1023/A:1006241729948
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    Purification and some properties of an α-amylase glucoamylase fusion protein from Saccharomyces cerevisiae (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 561-564 
    Details
    ISSN: 1573-0972
    Keywords: α-Amylase ; fusion protein ; glucoamylase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fusion gene containing the Bacillus subtilis α-amylase gene and Aspergillus awamori glucoamylase cDNA was expressed in Saccharomyces cerevisiae. The resulting bifunctional fusion protein having both α-amylase and glucoamylase activities secreted into the culture medium was purified to apparent homogeneity by affinity chromatography and gel filtration on Sephadex G-100. The enzyme had an apparent molecular mass of 150 kDa and showed an optimum pH and temperature of 6.0 and 60 °C, respectively. The main hydrolysis products from soluble starch were glucose and maltose.
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    URL: http://dx.doi.org/10.1023/A:1008961015119
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    Cloning and sequence analysis of Histoplasma capsulatum glucosamine-6-phosphate synthase gene fragment (1998)
    Springer
    Mycopathologia 142 (1998), S. 67-70 
    Details
    ISSN: 1573-0832
    Keywords: l-glutamine ; fructose-6-phosphate amidotransferase ; Candida albicans ; fungi ; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; systemic mycoses chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The 3' part of the glucosamine-6-phosphate synthase gene from Histoplasma capsulatum was PCR amplified using degenerate primers designed from the known glucosamine-6-phosphate synthase gene sequences, cloned and sequenced. The computer analysis of the 676 bp sequence revealed the presence of two introns. The identities of the deduced amino acid sequence to the corresponding Saccharomyces cerevisiae and Candida albicans fragment are 65 and 63.8%, respectively.
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    URL: http://dx.doi.org/10.1023/A:1006900321524
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    Effect of a Teucrium polium L. extract on the growth and fatty acid composition of Saccharomyces cerevisiae and Yarrowia lipolytica (1998)
    Springer
    Antonie van Leeuwenhoek 73 (1998), S. 195-198 
    Details
    ISSN: 1572-9699
    Keywords: growth inhibition ; fatty acid composition ; Saccharomyces cerevisiae ; Yarrowia lipolytica ; Teucrium polium L. extract
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Aqueous Teucrium polium extract slightly inhibits the growth of Saccharomyces cerevisiae (Ki=0.029 [g/l]-1) and Yarrovia lipolytica (Ki=0.061 [g/l]-1). However, this extract causes important changes in the unsaturation degree (Δ/mol) of the cellular lipids. It moreover favours the increase of the linolenic acid concentration and the decrease of the oleic one in both species.
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    URL: http://dx.doi.org/10.1023/A:1000673426077
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    Expression of HIV-1nefin yeast causes membrane perturbation and release of the myristylated Nef protein (1998)
    Springer
    Journal of biomedical science 5 (1998), S. 203-210 
    Details
    ISSN: 1423-0127
    Keywords: Acquired immunodeficiency syndrome ; Human immunodeficiency virus ; Nef protein ; Myristylation ; Membrane permeabilisation ; Saccharomyces cerevisiae ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial. During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet. This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies. Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies. These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV-1 infected cells.
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    URL: http://dx.doi.org/10.1007/BF02253470
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    Photoinhibitory damage is modulated by the rate of photosynthesis and by the photosystem II light-harvesting chlorophyll antenna size (1998)
    Springer
    Planta 205 (1998), S. 288-296 
    Details
    ISSN: 1432-2048
    Keywords: Key words: Chlorophyll antenna size ; D1 protein ; Dunaliella (photoinhibition) ; Electron transport ; Photoinhibition ; Photosystem II
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. We investigated the effect of photosynthetic electron transport and of the photosystem II (PSII) chlorophyll (Chl) antenna size on the rate of PSII photoinhibitory damage. To modulate the rate of photosynthesis and the light-harvesting capacity in the unicellular chlorophyte Dunaliella salina Teod., we varied the amount of inorganic carbon in the culture medium. Cells were grown under high irradiance either with a limiting supply of inorganic carbon, provided by an initial concentration of 25 mM NaHCO3, or with supplemental CO2 bubbled in the form of 3% CO2 in air. The NaHCO3-grown cells displayed slow rates of photosynthesis and had a small PSII light-harvesting Chl antenna size (60 Chl molecules). The half-time of PSII photodamage was 40 min. When switched to supplemental CO2 conditions, the rate of photodamage was retarded to a t1/2 = 70 min. Conversely, CO2-supplemented cells displayed faster rates of photosynthesis and a larger PSII light-harvesting Chl antenna size (500 Chl molecules). They also showed a rate of photodamage with t1/2 = 40 min. When depleted of CO2, the rate of photodamage was accelerated (t1/2  = 20 min). These results indicate that the in-vivo susceptibility to photodamage is modulated by the rate of forward electron transport through PSII. Moreover, a large Chl antenna size enhances the rate of light absorption and photodamage and, therefore, counters the mitigating effect of forward electron transport. We propose that under steady-state photosynthesis, the rate of light absorption (determined by incident light intensity and PS Chl antenna size) and the rate of forward electron transport (determined by CO2 availability) modulate the oxidation/reduction state of the primary PSII acceptor QA, which in turn defines the low/high probability for photodamage in the PSII reaction center.
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    URL: http://dx.doi.org/10.1007/s004250050323
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    Gene-conversion tract directionality is influenced by the chromosome environment (1998)
    Springer
    Current genetics 34 (1998), S. 269-279 
    Details
    ISSN: 1432-0983
    Keywords: Key words Double-strand breaks ; Heteroduplex DNA ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Spontaneous and double-strand break (DSB)-induced gene conversion in Saccharomyces cerevisiae was assayed using non-tandem chromosomal direct repeat crosses and plasmid × chromosome crosses. Each cross involved identical ura3 alleles marked with phenotypically silent restriction fragment length polymorphic (RFLP) mutations at approximately 100-bp intervals. DSBs introduced in vivo at HO sites in one allele stimulated recombination to Ura+ by more than two orders of magnitude. Spontaneous gene-conversion products were isolated from a related strain lacking a functional HO nuclease gene. The multiple markers did not appear to influence the frequency of direct repeat deletions for spontaneous or DSB-induced events. DSB-induced conversion reflected efficient mismatch repair of heteroduplex DNA. Conversion frequencies of equidistant markers on opposites sides of the DSB were similar in the direct repeat cross. In contrast, markers 5′ of the DSB (promoter-proximal) converted more often than 3′ markers in plasmid × chromosome crosses, a possible consequence of crossing-over associated with long conversion tracts. With direct repeats, bidirectional tracts (extending 5′ and 3′ of the DSB) occurred twice as often as in a plasmid × chromosome cross in which DSBs were introduced into the plasmid-borne allele. A key difference between the direct-repeat and plasmid×chromosome crosses is that the ends of a broken plasmid are linked, whereas the ends of a broken chromosome are unlinked. We tested whether linkage of ends influenced tract directionality using a second plasmid × chromosome cross in which DSBs were introduced into the chromosomal allele and found few bidirectional tracts. Thus, chromosome environment, but not linkage of ends, influences tract directionality. The similar tract spectra of the two plasmid × chromosome crosses suggest that similar mechanisms are involved whether recombination is initiated by DSBs in plasmid or chromosomal alleles.
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    URL: http://dx.doi.org/10.1007/s002940050396
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    The Saccharomyces cerevisiae gene PSO5/RAD16 is involved in the regulation of DNA damage-inducible genes RNR2 and RNR3 (1998)
    Springer
    Current genetics 34 (1998), S. 124-127 
    Details
    ISSN: 1432-0983
    Keywords: Key wordsPSO5/RAD16 ; Saccharomyces cerevisiae ; Nucleotide excision repair ; Oxidative stress ; Ribonucleotide reductase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The expression of β-galactosidase from DNA damage-inducible RNR2-lacZ and RNR3-lacZ fusion constructs was compared in wild-type (WT) and pso5/rad16 mutant strains after treatment with five mutagens/oxidative stressors. While exposure to the mutagens UVC, 4NQO and H2O2 induced expression of the RNR2-lacZ and RNR3-lacZ fusion constructs in two WT strains, treatment with the two oxidative stressors tBOOH and paraquat did not. In the pso5-1 mutant induction of RNR2-lacZ was largely reduced after UVC and H2O2 while there was no significant induction of β-galactosidase expression after 4NQO treatment for this construct. For RNR3-lacZ there was strongly reduced expression of pso5-1 after UVC and 4NQO while H2O2 failed to induce expression of β-galactosidase. In the WT strains the ranking of the inducing power of the mutagens at 90% survival (as measured in the pso5-1 mutant) was 4NQO〉UVC〉H2O2. Though the WT strains were clearly more resistant that the pso5-1 mutant to the two oxidative stressors paraquat and tBOOH, these substances failed to significantly enhance expression of the RNR2-lacZ and RNR3-lacZ fusion constructs in both the WT and the pso5-1 mutant. Our data suggest that Pso5p/Rad16p has a function in the signal transducing pathway controlling DNA damage-inducible components of nucleotide excision repair.
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    URL: http://dx.doi.org/10.1007/s002940050376
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    Azf1p is a nuclear-localized zinc-finger protein that is preferentially expressed under non-fermentative growth conditions in Saccharomyces cerevisiae (1998)
    Springer
    Current genetics 34 (1998), S. 287-296 
    Details
    ISSN: 1432-0983
    Keywords: Key words Zinc-finger protein ; Nuclear localization ; Immuno electron microscopy ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In previous studies the AZF1 gene has been identified as a second high-copy number suppressor for a special mutant of the gene for the mitochondrial core enzyme of RNA polymerase. The first high-copy number suppressor of this mutant turned out to be the specificity factor MTF1 for mitochondrial transcription. Up to now, the influence of AZF1 on mitochondrial transcription, its precise localization in the cell and the regulation of its expression has not been determined. The putative protein contains a long stretch of poly-asparagine amino acids and a typical zinc-finger domain for DNA binding. These characteristic structural features were used to create the abbreviation AZF1 (Asparagine-rich Zinc Finger protein). An initial computer analysis of the sequence gave no conclusive results for the presence of a mitochondrial import sequence or a typical nuclear-targeting sequence. A recent more-detailed analysis identified a possible nuclear localization signal in the middle of the protein. Disruption of the gene shows no effect on plates with glucose-rich medium or glycerol. In this report a specific polyclonal antibody against Azf1p was prepared and used in cell-fractionation experiments and in electron-microscopic studies. Both of these clearly demonstrate that the AZF1 protein is localized exclusively in the nucleus of the yeast cell. Northern analysis for the expression of the AZF1 messenger RNA under different growth conditions was therefore performed to obtain new insights into the regulation of this gene. Together with the respective protein-expression analysis these data demonstrate that Azf1p is preferentially synthezised in higher amounts under non-fermentable growth conditions. Over-expression of Azf1p in the yeast cell does not influence the expression level of the mitochondrial transcription factor Mtf1p, indicating that the influence of Azf1p on the suppression of the special mitochondrial RNA polymerase mutant is an indirect one. Subcellular investigation of the deletion mutant by electron microscopy identifies specific ultrastructural cell-division defects in comparison to the wild-type.
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    URL: http://dx.doi.org/10.1007/s002940050398
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    Mitotic recombination and localized DNA double-strand breaks are induced after 8-methoxypsoralen and UVA irradiation in Saccharomyces cerevisiae (1998)
    Springer
    Current genetics 34 (1998), S. 30-42 
    Details
    ISSN: 1432-0983
    Keywords: Key words Mitotic recombination ; DNA double-strand breaks ; Saccharomyces cerevisiae ; 8-Methoxypsoralen plus UVA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mitotic recombination within the ARG4 gene of Saccharomyces cerevisiae was analysed after treatment of cells with the recombinogenic agent 8-methoxypsoralen (8-MOP) plus UVA. The appearance of DNA double-strand breaks (DSBs) in the ARG4 region during post-treatment incubation was also tested. The results obtained after 8-MOP plus UVA treatment indicate that in mitotic cells: (1) recombination at the ARG4 locus is increased 30 – 500 fold per survivor depending on the strains and the doses employed, (2) the increase of recombination results essentially from gene conversion events which involve the RV site located in the 5′ region of the ARG4 gene twice as often as the Bgl site at the 3′ end, (3) depending on 8-MOP/UVA dose, ectopic gene conversion is associated with reciprocal translocation, (4) DSBs occur preferentially in the ARG 5′ region during post-treatment incubation, as well as in other intergenic regions containing both promoters or/and terminators of transcription, and (5) changes in sequence content in the 5′ region of ARG4, which influences positions and frequencies of DSBs formed during repair, are correlated with a modification of the local chromatin structure.
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    Reciprocal translocation at duplicated RPL2 loci might cause speciation of Saccharomyces bayanus and Saccharomyces cerevisiae (1998)
    Springer
    Current genetics 33 (1998), S. 345-351 
    Details
    ISSN: 1432-0983
    Keywords: Key wordsSaccharomyces bayanus ; Saccharomyces cerevisiae ; Translocation ; Speciation ; Duplicated gene ; RPL2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract By a genomic comparison of two sibling yeasts, Saccharomyces bayanus and S. cerevisiae, we previously demonstrated that chromosomes II and IV of S. cerevisiae were rearranged into chromosomes 12 and 14 of S. bayanus or vice versa. In the present study we have delimited the translocation break sites in chromosomes II and IV by Southern hybridization using DNA fragments of S. cerevisiae cosmid clones as probes. The results suggest that the reciprocal translocation of chromosomes II and IV had occurred at duplicated RPL2 loci. Furthermore, the translocation sites in S. bayanus were confirmed by the cloning and sequence analysis of the regions flanking RPL2 loci. Several genes in the regions flanking the RPL2 loci were present in the order expected for a translocation at these loci between the two species. These results indicated that the reciprocal translocation between chromosomes II and IV was generated by homologous recombination at duplicated RPL2 loci on the two chromosomes. Therefore, we propose that duplicated genes or duplicated regions play an important role in altering genomic organization during the speciation of S. bayanus and S. cerevisiae.
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    Functional analysis of upstream activating elements in the promoter of the FBP1 gene from Saccharomyces cerevisiae (1998)
    Springer
    Current genetics 33 (1998), S. 406-411 
    Details
    ISSN: 1432-0983
    Keywords: Key words Fructose-1 ; 6-bisphosphatase ; Catabolite repression ; Gluconeogenesis ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have investigated the effect of different carbon sources and of different mutations on the capacity of two elements, UAS1 and UAS2, from the promoter of the FBP1 gene to form specific DNA-protein complexes and to activate expression of a reporter gene. The complexes are observed with nuclear extracts from yeast derepressed on glycerol or ethanol. When hxk2 mutants are grown on glucose the nuclear extracts are able to complex UAS1 but not UAS2, while for wild-type cells grown on galactose only the complex with UAS2 is formed. In contrast, in vivo the operation of both UASs is high in ethanol, moderate to low in glycerol, and negligible in galactose; no expression is observed in glucose even in a hxk2 background. There is no effect of a MIG1 deletion, either in the formation of DNA-protein complexes or on the expression of reporter genes.
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    Molecular and biochemical analysis of Saccharomyces cerevisiae cox1 mutants (1998)
    Springer
    Current genetics 34 (1998), S. 138-145 
    Details
    ISSN: 1432-0983
    Keywords: Key words Cytochrome c oxidase ; Saccharomyces cerevisiae ; Complex assembly
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report on the molecular and biochemical analysis of a set of 13 respiratory deficient mutants of Saccharomyces cerevisiae which are specifically altered in COX1, the gene encoding the subunit Cox1p of cytochrome c oxidase. DNA sequence analysis shows that three are due to frameshift mutations, two to nonsense mutations, and eight to missense mutations. All, except the missense mutant S157L, have impaired electron transfer and respiratory activity. Analysis of the mitochondrial translation products shows that when Cox1p is absent, Cox2p and Cox3p are still synthesized. In the missense mutants, the steady state levels in the mitochondrial membranes of the three mitochondrially encoded subunits Cox1p, Cox2p and Cox3p and the nuclear-encoded subunit Cox4p are reduced. In the frameshift and nonsense mutants, Cox1p is absent and Cox2p, Cox3p and Cox4p are considerably decreased or undetectable. A comparison of the steady state levels of Cox1p through Cox4p in the COX1, COX2, COX3 and COX4 mutants shows the interdependance of the accumulation of these four subunits in the mitochondrial membranes.
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    Activation of the H+-ATPase in the plasma membrane of cells of Saccharomyces cerevisiae grown under mild copper stress (1998)
    Springer
    Archives of microbiology 171 (1998), S. 6-12 
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    ISSN: 1432-072X
    Keywords: Key words Plasma membrane H+-ATPase ; Saccharomyces cerevisiae ; Copper stress ; PMA1 ; PMA2 ; Gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cells of Saccharomyces cerevisiae exibited a more active plasma membrane H+-ATPase during growth in media supplemented with CuSO4 concentrations equal to or below 1 mM than did cells cultivated in the absence of copper stress. Maximal specific activities were found with 0.5 mM CuSO4. ATPase activity declined when cells were grown with higher concentrations up to 1.5 mM (the maximal concentration that allowed growth), probably due to severe disorganization of plasma membrane. Cu2+-induced maximal activation was reflected in an increase of V max (approximately threefold) and in the slight decrease of the K m for MgATP (from 0.93 ± 0.13 to 0.65 ± 0.16 mM). The expression of the gene encoding the essential plasma membrane ATPase (PMA1) was reduced with a dose-dependent pattern in cells grown with inhibitory concentrations of copper, while the weakly expressed PMA2 gene promoter was moderately more efficient in cells cultivated under mild copper stress (1.5-fold maximal activation). ATPase was activated by copper despite the slightly lower content of ATPase protein in the plasma membrane of Cu2+-grown cells and the powerful inhibitory effect of Cu2+ in vitro.
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    Yeast mitochondrial metabolism: From in vitro to in situ quantitative study (1998)
    Springer
    Molecular and cellular biochemistry 184 (1998), S. 67-79 
    Details
    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; spheroplast ; permeabilization ; mitochondria ; oxidative phosphorylation ; porin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In this work, we first compared yeast mitochondrial oxidative metabolism at different levels of organization: whole cells (C), spheroplasts (S), permeabilized spheroplasts (PS) or isolated mitochondria (M). At present, S are more suitable for use than C for biochemical techniques such as fast extraction of metabolises and permeabilization. We show here that respiratory rates of S with various substrates are similar to C, which demonstrate that they are adapted to yeast bioenergetic studies. It appeared from ethanol metabolism ± NAD++ or NADH respiratory rates on PS that ethanol metabolism was largely cytosolic; moreover, the activity of NADH dehydrogenase was lesser in the case of PS than in S. By comparing PS and M, the biggest difference concerned the respiratory rates of pyruvate and pyruvate-malate, which were much lower for M. Thus mitochondria preparation caused an unidentified loss involved directly in pyruvate metabolism. When the respiratory rate was lowered as a consequence of a high kinetic control of oxidative activity upstream from the respiratory chain, a similar correlation between the increase in ATP/O and decrease in respiratory rate was observed. So, the intrinsic uncoupling of proton pumps is not a particularity of M. Secondly, we demonstrate the existence of a mechanism of retarded diffusion in yeast similar to that already observed in permeabilized mammalian cells for ADP. Such a mechanism also occurs in yeast for several respiratory substrates: the K0.5 for each substrate toward the respiration rate in PS always exceeds that for M. It is proposed that such a discrepancy is due to a restriction of metabolite movement across the outer mitochondrial membrane in permeabilized cells, i.e. regulation of the substrate permeability through porin channels. In the porin-deficient yeast mutant, the K0.5 for NADH is not significantly different in either M or PS and is comparable to that of the parent strain PS. This result confirms that this retarded diffusion is essentially due to the opening-closing of the porin channel.
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    URL: http://dx.doi.org/10.1023/A:1006830810440
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    Comparison of biochemical effects produced by calcium ions and by monomers of polyacrylamide (acrylamide and bisacrylamide) on strains of Saccharomyces cerevisiae used for production of chiral synthons (1998)
    Springer
    Molecular and cellular biochemistry 178 (1998), S. 33-40 
    Details
    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; NAD(P)H ; calcium ions ; cells immobilization ; oxygen consumption ; biotransformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The biochemical behaviour of four commercial strains of Saccharomyces cerevisiae was studied in the presence of calcium ions, acrylamide and bisacrylamide. Calcium ions at a concentration of 300 µM induced an increase of NAD(P)+ reduction in commercial Turkish and American strains, while in Chilean and Brazilian commercial strains, it diminished NAD(P)+ reduction. On the other hand, polyacrylamide monomers (acrylamide and bisacrylamide) induced a decrease of NAD(P)+ reduction in all strains studied in this paper. When membrane potential (ΔΨ) and oxygen consumption were measured in the presence of polyacrylamide monomers, a decrease of both was observed in all strains studied.
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    URL: http://dx.doi.org/10.1023/A:1006834404049
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    The use of molecular modelling in the understanding of configurational specificity (R or S) in asymmetric reactions catalyzed by Saccharomyces cerevisiae or isolated dehydrogenases (1998)
    Springer
    Molecular and cellular biochemistry 178 (1998), S. 27-31 
    Details
    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; microorganisms ; dehydrogenases ; acetoacetate ; molecular modelling ; enantiomeric excess ; biotransformation ; baker's yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This method gives a general ideal how to use crystallographic information of enzymes to understand reactions catalyzed by these biocatalysts, commonly used by biochemists to produce chiral products. The interactions of three acetoacetic esters with the enzymes L-lactate dehydrogenase and alcohol dehydrogenase were studied through molecular modelling computer program. These artificial substrates have been widely used to produce chiral synthons. Through this methodology it was possible to understand the conformational specificity of these enzymes with respect to the products and how these enzymes can be inhibited by modifying the structures of the artificial substrates. Also, it was possible to predict whether some type of artificial substrate will suffer reduction by cells that contain these dehydrogenases and what kind of configuration (R or S) the final product will have.
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    URL: http://dx.doi.org/10.1023/A:1006831902849
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  • 89
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    Thermoluminescence studies on the facultative crassulacean-acid- metabolism plant Mesembryanthemum crystallinum L. (1998)
    Springer
    Planta 205 (1998), S. 587-594 
    Details
    ISSN: 1432-2048
    Keywords: Key words: Crassulacean acid metabolism ; Mesembry-anthemum ; Photosystem II ; Thermoluminescence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. Thermoluminescence (TL) signals were measured from leaves of the facultative CAM (crassulacean acid metabolism) plant Mesembryanthemum crystallinum L.. Following the induction of CAM by salt treatment, a TL band at 46 °C was induced, which was charged by a single-turnover flash. The intensity of the 46 °C-band depends on the number of excitation flashes and oscillates with a period of four. A similar band was induced in C3 plants by far-red illumination. Under CAM conditions, the intensity of the 46 °C-band underlies a diurnal rhythm. The maximal intensity of the 46 °C-band is observed in the morning after onset of the light and in the evening. At around 12 a.m. it is suppressed. The intensity of the 46 °C-band relates to diurnal changes in the ratio of dihydroxy acetone phosphate/3-phosphoglycerate (DHAP/PGA) which is an indicator of the energy status of the chloroplast. During high-intensity illumination, the 46 °C-band disappears, but it is restored in the dark. We propose that the 46 °C-band is an indicator of the metabolic state of the leaf, originating from photosystem II centres initially in the S2(S3)QB oxidation state, in which the electron acceptor QB becomes reduced either by reverse electron flow or reduction of the plastoquinone pool via an NAD(P)H plastoquinone oxidoreductase. We present evidence that the redox state of the electron-transport chain is different under conditions of CAM compared to C3 metabolism and that changes induced by CAM can be monitored by measuring the amplitude of the 46 °C-band after flash excitation.
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    URL: http://dx.doi.org/10.1007/s004250050360
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  • 90
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    The Saccharomyces cerevisiaeSOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane (1998)
    Springer
    Molecular genetics and genomics 257 (1998), S. 635-640 
    Details
    ISSN: 1617-4623
    Keywords: Key words Mitochondrial protein sorting ; Processing of Cox2 ; Kluyveromyces lactis ; Leishmania major ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28° C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane.
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    URL: http://dx.doi.org/10.1007/s004380050691
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  • 91
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    Growth-regulated formation of heteromeric complexes of the centromere and promoter factor, Cbf1p, in yeast (1998)
    Springer
    Molecular genetics and genomics 260 (1998), S. 417-425 
    Details
    ISSN: 1617-4623
    Keywords: Key words Centromere and promoter factor 1 (Cpf1p) ; Protein-protein interaction ; Saccharomyces cerevisiae ; Environmental adaptations ; Transcriptional activation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transcriptional regulation of the yeast cytochrome c 1 gene (CYT1) in response to oxygen and carbon source is mediated by Hap1p and the Hap2 complex. Furthermore, the centromere-binding factor 1 (Cbf1p) associates with the CYT1 upstream region (UASCYT1), but its direct activation potential is insignificant. The possible role of Cbf1p as a modulator of transcriptional adaptation to changes in nutritional conditions was examined. In electrophoretic mobility shift assays (EMSA) using yeast nuclear extracts, Cbf1p was found to exist as homo- and heterodimers of processed subforms of 54 and 37 kDa. An additional 18-kDa version was the only species found in anaerobic cells grown under an atmosphere of purified nitrogen, but not when CO2 was used to establish anaerobiosis. All three dimers of the 37 and 54 kDa versions of Cbf1p that occurred in oxidatively growing cells gave rise to hetero-oligomeric complexes containing other as yet unidentified protein(s). Complex formation was not observed with extracts from cultures grown on high levels of glucose and was dependent on pre-assembly in the absence of target DNA. Pre-treatment with alkaline phosphatase enhanced formation of these higher-order complexes. The C-terminal 18-kDa segment of Cbf1p, which can undergo dimerization and bind DNA, does not induce supershifts after preincubation and is not influenced by dephosphorylation. We propose that the N-terminal domain is subject to carbon source- or growth-dependent phosphorylation/dephosphorylation events that result in differential recruitment of additional factors to promoters of genes that encode proteins required for non-fermentative growth.
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    URL: http://dx.doi.org/10.1007/s004380050912
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  • 92
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    PsbY, a novel manganese-binding, low-molecular-mass protein associated with photosystem II (1998)
    Springer
    Molecular genetics and genomics 260 (1998), S. 56-68 
    Details
    ISSN: 1617-4623
    Keywords: Key words PsbY ; Photosystem II ; Gene duplication ; Polyprotein ; Topogenesis ; L-Arginine metabolizing enzyme
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We describe two related manganese-binding polypeptides with L-arginine metabolizing enzyme activity that can be detected as distinct components (designated PsbY-A1 and PsbY-A2, previously called L-AME) in membranes containing Photosystem II (PS II) from spinach. The polypeptides are bitopic and appear to exist in a heterodimeric form, but only in the chlorophyll a/b lineage of plants. Both proteins are encoded in the nucleus. In spinach and in Arabidopsis thaliana they are both derived from a single-copy gene (psbY) that is translated into a precursor polyprotein of approximately 20 kDa. The processing of the polyprotein is complex and includes at least four cleavage steps. Both polypeptides are exposed N-terminally to the lumenal and C-terminally to the stromal face of the thylakoid membrane.
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    URL: http://dx.doi.org/10.1007/s004380050870
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    Growth inhibition of Saccharomyces cerevisiae by the immunosuppressant leflunomide is due to the inhibition of uracil uptake via Fur4p (1998)
    Springer
    Molecular genetics and genomics 260 (1998), S. 102-107 
    Details
    ISSN: 1617-4623
    Keywords: Key words Immunosuppressant ; Uracil permease ; FUR4 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The immunosuppressant leflunomide inhibits cytokine-stimulated proliferation of lymphoid cells in vitro and also inhibits the growth of the eukaryotic microorganism Saccharomyces cerevisiae. To elucidate the molecular mechanism of action of the drug, two yeast genes which suppress the anti-proliferative effect when present in multiple copies were cloned and designated MLF1 and MLF2 for multicopy suppressor of leflunomide sensitivity. DNA sequencing analysis revealed that the MLF1 gene is identical to the FUR4 gene, which encodes a uracil permease and functions to import uracil efficiently. The MLF2 was found to be identical to the URA3 gene. Excess exogenous uracil also overcomes the anti-proliferative effect of leflunomide on yeast cells. Uracil prototrophy also conferred resistance to leflunomide. Uracil uptake was inhibited by leflunomide. Thus, the growth inhibition by leflunomide seen in a S. cerevisiae ura3 auxotroph is due to the inhibition of the entry of exogenous uracil via the Fur4 uracil permease.
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    URL: http://dx.doi.org/10.1007/s004380050875
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    Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing (1998)
    Springer
    Molecular genetics and genomics 260 (1998), S. 232-241 
    Details
    ISSN: 1617-4623
    Keywords: Key words Cell cycle ; mRNA splicing ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export.
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    URL: http://dx.doi.org/10.1007/s004380050891
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    A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene (1998)
    Springer
    Molecular genetics and genomics 258 (1998), S. 148-155 
    Details
    ISSN: 1617-4623
    Keywords: Key words Protein kinase C ; Signal transduction ; Transposon mutagenesis ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We employed the constitutive BCK1-20 allele of the gene for the MAP kinase kinase kinase (MAPKKK) in the yeast Pkc signal transduction pathway to develop a genetic screen for mutants in genes encoding upstream components. Transposon mutagenesis yielded a mutant that was completely dependent on the active allele in the absence of osmotic stabilization. The transposon had integrated at the yeast SLG1 (HCS77) locus. This gene encodes a putative membrane protein. Haploid slg1 deletion strains are sensitive to caffeine, as expected for mutants in the Pkc pathway, as well as a variety of other drugs. The response to elevated temperatures and the dependence on osmotic stabilization depends on the genetic background. Thus, in the strain used for mutagenesis, disruption of SLG1 causes the cells to become non-viable in the absence of osmotic stabilization at both 30° C and 37° C. In a different genetic background this phenotype was not observed. Sensitivity of the haploid deletion mutants to caffeine can be partially suppressed by overexpression of genes for other components of the Pkc pathway, such as PKC1, SLT2, ROM2, and STE20. In addition, a SLG1-lacZ reporter construct shows higher expression in the presence of caffeine or magnesium chloride in a wild-type diploid background.
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    URL: http://dx.doi.org/10.1007/s004380050717
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    DNA replication is completed in Saccharomyces cerevisiae cells that lack functional Cdc14, a dual-specificity protein phosphatase (1998)
    Springer
    Molecular genetics and genomics 258 (1998), S. 437-441 
    Details
    ISSN: 1617-4623
    Keywords: Key words Dual-specificity phosphatase ; DNA synthesis ; Telophase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Cdc14 protein encodes a dual-specificity protein phosphatase which functions in late mitosis, and considerable genetic evidence suggests a role in DNA replication. We find that cdc14 mutants arrested in late mitosis maintain persistent levels of mitotic kinase activity, suggesting that Cdc14 controls inactivation of this kinase. Overexpression of Sic1, a cyclin-dependent protein kinase inhibitor, is able to suppress telophase mutants such as dbf2, cdc5 and cdc15, but not cdc14. It does, however, force cdc14-arrested cells into the next cell cycle, in which an apparently normal S phase occurs as judged by FACS and pulsed-field gel electrophoretic analysis. Furthermore, in a promoter shut-off experiment, cells lacking Cdc14 appear to carry out a normal S phase. Thus Cdc14 functions mainly in late mitosis and it has no essential role in S phase.
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    URL: http://dx.doi.org/10.1007/s004380050753
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    A physical assay for detection of early meiotic recombination intermediates in Saccharomyces cerevisiae (1998)
    Springer
    Molecular genetics and genomics 258 (1998), S. 512-520 
    Details
    ISSN: 1617-4623
    Keywords: Key words Homologous recombination ; Double-strand breaks ; Recombination intermediate ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In most eukaryotic organisms, recombination events leading to exchanges between homologous chromosomes link the homologs in a manner that allows their proper attachment to the meiotic spindle. In the yeast Saccharomyces cerevisiae these exchanges are initiated in early prophase as double-strand breaks in the DNA. These breaks are processed through a series of intermediates to yield mature crossovers late in prophase. The following experiments were designed to monitor the appearance of the earliest recombinant DNA strands formed in this process. A polymerase chain reaction assay was devised that allows the detection of recombinant strands at a known initiation site for meiotic recombination. The time and rate of appearance of recombinant strands was found to coincide with commitment to recombination, demonstrating that DNA strands bearing sequences from both parental chromosomes are rapidly formed after the initiation of meiotic recombination.
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    URL: http://dx.doi.org/10.1007/s004380050762
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    Accumulation of β-tubulin-containing fibres in the cortical domain ofSaccharomyces cerevisiae spheroplasts (1998)
    Springer
    Protoplasma 202 (1998), S. 11-16 
    Details
    ISSN: 1615-6102
    Keywords: GeneTUB2 ; Saccharomyces cerevisiae ; Antibody TU-14 ; Cortical β-tubulin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The distribution of microtubules inSaccharomyces cerevisiae was studied with the monoclonal antibody (mab) TU-14 against β-tubulin. Immunoblotting and immunoprecipitation experiments with a strain overexpressing Tub2p confirmed that the mab TU-14 specifically recognized Tub2p. By immunofluorescence microscopy, the mab TU-14 attached to all known tubulin structures labelled with the standard polyclonal anti-β-tubulin antibody 206-1. In addition, the mab TU-14 revealed cortical patches in wild-type cells and an abundant network of fibres in the cortex of spheroplasts cultivated in nutrient medium. These cortical fibres seemed to be specific to spheroplasts and suggest that the accumulated Tub2p is predominantly associated with the plasma membrane.
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    URL: http://dx.doi.org/10.1007/BF01280870
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    Characterization of oligosaccharides from an antigenic mannan of Saccharomyces cerevisiae (1998)
    Springer
    Glycoconjugate journal 15 (1998), S. 815-822 
    Details
    ISSN: 1573-4986
    Keywords: Saccharomyces cerevisiae ; oligosaccharide structure ; antigenic glycoprotein ; mannan ; allergens
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Mannans of the yeast Saccharomyces cerevisiae have been implicated as containing the allergens to which bakers and brewers are sensitive and also the antigen recognized by patients with Crohn's disease. A fraction of S. cerevisiae mannan, Sc500, having high affinity for antibodies in Crohn's patients has been characterized by NMR spectroscopy followed by fragmentation using alkaline elimination, partial acid hydrolysis and acetolysis. The released oligosaccharides were separated by gel filtration on a Biogel P4 column and analyzed by fluorescence labeling, HPLC and methylation analysis. The relationship between structure and antigen activity was measured by competitive ELISA. The antigenic activity of the original high molecular weight mannan could be ascribed to terminal Manα1→3Manα1→2 sequences which are rarely found in human glycoproteins but were over-represented in Sc500 compared to other yeast mannans.
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    URL: http://dx.doi.org/10.1023/A:1006968117252
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    The photosystem II subunit CP29 can be phosphorylated in both C3 and C4 plants as suggested by sequence analysis (1998)
    Springer
    Plant molecular biology 36 (1998), S. 11-22 
    Details
    ISSN: 1573-5028
    Keywords: chlorophyll a/b protein ; CP29 ; Phosphorylation ; Photosystem II ; cold stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The CP29 subunit of Photosystem II is reversibly phosphorylated in Zea mays upon exposure to high light in the cold (Bergantino et al., J Biol Chem 270 (1995) 8474–8481). This phenomenon was previously proposed to be restricted to C4 plants. We present the complete sequence of the CP29 protein, deduced from a maize Lhcb4 cDNA clone, and its comparison with the previously known Lhcb4 sequences of two C3 plants: Hordeum vulgare and Arabidopsis thaliana. Despite the relatively low degree of homology in their amino-terminal region, i.e. the part of the molecule which is phosphorylated in maize, the three polypeptides conserve consensus sequences for the site of phosphorylation. We proved by immunoblotting and 33P-labelling that the same post-translational modification occurs in barley. Being thus common to C3 and C4 plant species, the phosphorylation of this minor antenna complex of Photosystem II appears now as a widespread phenomenon, possibly part of the phosphorylation cascade which signals the redox status of the plastoquinone to the nuclear transcription apparatus. Arabidopsis plants do not show phosphorylation of CP29 in the same conditions, but other low-molecular-weight phosphoproteins, whose role need to be elucidated, become evident.
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    URL: http://dx.doi.org/10.1023/A:1005904527408
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