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  • 1
    ISSN: 1432-0983
    Keywords: Hansenula polymorpha ; Peroxisomes ; Organelle biogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the methylotrophic yeast Hansenula polymorpha, approximately 25% of all methanol-utilization-defective (Mut-) mutants are affected in genes required for peroxisome biogenesis (PER genes). Previously, we reported that one group of per mutants, termed Pim-, are characterized by the presence of a few small peroxisomes with the bulk of peroxisomal enzymes located in the cytosol. Here, we describe a second major group of per mutants that were observed to be devoid of any peroxisome-like structure (Per-). In each Per- mutant, the peroxisomal methanol-pathway enzymes alcohol oxidase, catalase and dihydroxyacetone synthase were present and active but located in the cytosol. Together, the Pim- and Per- mutant collections involved mutations in 14 different PER genes. Two of the genes, PER5 and PER7, were represented by both dominant-negative and recessive alleles. Diploids resulting from crosses of dominant per strains and wild-type H. polymorpha were Mut- and harbored peroxisomes with abnormal morphology. This is the first report of dominant-negative mutations affecting peroxisome biogenesis.
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  • 2
    ISSN: 1432-072X
    Keywords: Hansenula polymorpha ; Peroxisomes ; Alcohol oxidase ; Protein assemblage ; FAD ; Cyanide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The fate of alcohol oxidase (AO) in chemostatgrown cells of Hansenula polymorpha, after its inactivation by KCN, was studied during subsequent cultivation of the cyanide-treated cells in fresh methanol media. Biochemical experiments showed that the cyanide-induced inactivation of AO was due to the release of flavin adenine dinucleotide (FAD) from the holo enzyme. However, dissociation of octameric AO into subunits was not observed. Subsequent growth of intact cyanide-treated cells in fresh methanol media was paralelled by proteolytic degradation of part of the peroxisomes present in the cells. The recovery of AO activity, concurrently observed in these cultures, was accounted for by synthesis of new enzyme protein. Reactivation of previously inactivated AO was not observed, even in the presence of FAD in such cultures. Newly synthesized AO protein was incorporated in only few of the peroxisomes present in the cells. 31P nuclear magnetic resonance (NMR) studies showed that cyanide-treatment of the cells led to a dissipation of the pH gradient across the peroxisomal membrane. However, restoration of this pH gradient was fast when cells were incubated in fresh methanol medium after removal of the cyanide.
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  • 3
    ISSN: 1432-0983
    Keywords: Peroxisomes ; Protein import ; Saccharomyces cerevisiae ; Hansenula polymorpha
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The DAS gene of Hansenula polymorpha was expressed in Saccharomyces cerevisiae under the control of different promoters. The heterologously synthesized dihydroxyacetone synthase (DHAS), a peroxisomal enzyme in H. polymorpha, shows enzymatic activity in baker's yeast. The enzyme was imported into the peroxisomes of S. cerevisiae not only under the appropriate physiological conditions for peroxisome proliferation (oleic acid media), but also in glucose-grown cells where it induced the enlargement of the few peroxisomes present. This growth process was not accompanied by an increase in the number of microbodies, which suggests a separate control mechanism for peroxisomal proliferation.
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  • 4
    ISSN: 1432-0983
    Keywords: Transformation ; Electroporation ; Methylotrophic yeasts ; Hansenula polymorpha ; Pichia methanolica
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A highly-efficient method for transformation of the methylotrophic yeast Hansenula polymorpha has been developed. Routinely, transformation frequencies of up to 1.7×106/μg plasmid DNA were obtained by applying an electric pulse of the exponential decay type of 7.5 kV/cm to a highly-concentrated cell mixture during 5 ms. Efficient transformation was dependent on: (1) pretreatment of the cells with the reducing agent dithiotreitol, (2) the use of sucrose as an osmotic stabilizer in an ionic electroporation buffer, and (3) the use of cells grown to the mid-logarithmic phase. Important parameters for optimizing the transformation frequencies were field strength, pulse duration, and cell concentration during the electric pulse. In contrast to electrotransformation protocols described for Saccharomyces cerevisiae and Candida maltosa, transformation frequencies (transformants per μg DNA) for H. polymorpha remained high when large amounts (up to 10μg) of plasmid DNA were added. This feature renders this procedure pre-eminently advantageous for gene cloning experiments when high numbers of transformants are needed.
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  • 5
    ISSN: 1432-072X
    Keywords: Microbodies ; Biogenesis ; Yeasts ; Hansenula polymorpha ; Candida utilis ; Candida boidinii
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase. However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.
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  • 6
    ISSN: 1432-072X
    Keywords: Hansenula polymorpha ; Peroxisomes ; Peroxisome function ; Peroxisome-deficient mutant ; Methanol metabolism ; Continuous cultures ; Mixed-substrates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorphoa. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wild-type (WT) levels, this mutant is unable to grow on methanol as a sole carbon and energy source. Addition of methanol to glucose-limited (SR=12.5mM) chemostat cultures of the PER mutant only resulted in an increase in yield when small amounts were used (up to 22.5 mM). At increasing amounts however, a gradual decrease in cell density was observed which, at 80 mM methanol in the feed, had dropped below the original value of the glucose-limited culture. This reduction in yield was not observed when increasing amounts of formate instead of methanol were used as supplements for the glucose-limited mutant culture and also not in WT cells, used as control in these experiments. The effect of addition of methanol to a glucose-limited PER culture was also studied in the transient state during adaptation of the cells to methanol. The enzyme patterns obtained suggested that the ultimate decrease in yield observed at enhanced methanol concentrations was due to an inefficient methanolmetabolism as a consequence of the absence of peroxisomes. The absence of intact peroxisomes results in two major problems namely i) in H2O2-metabolism, which most probably is no longer mediated by catalase and ii) the inability of the cell to control the fluxes of formaldehyde, generated from methanol. The energetic consequences of this metabolism, compared to the WT situation with intact peroxisomes, are discussed.
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  • 7
    ISSN: 1432-072X
    Keywords: Peroxisome ; Degradation ; Autophagy ; Catabolite inactivation ; Alcohol oxidase ; Catalase ; Cytochemical staining ; Ultracryotomy ; Hansenula polymorpha
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Inactivation of peroxisomal enzymes in the yeast Hansenula polymorpha was studied following transfer of cells into cultivation media in which their activity was no longer required for growth. After transfer of methanol-grown cells into media containing glucose — a substrate that fully represses alcohol oxidase synthesis — the rapid inactivation of alcohol oxidase and catalase was paralleled by a disappearance of alcohol oxidase and catalase protein. The rate and extent of this inactivation was dependent upon conditions of cultivation of cells prior to their transfer. This carbon catabolite inactivation of alcohol oxidase was paralleled by degradation of peroxisomes which occurred by means of an autophagic process that was initiated by the formation of a number of electron-dense membranes around the organelles to be degraded. Sequestration was confined to peroxisomes; other cell-components such as ribosomes were absent in the sequestered cell compartment. Also, cytochemically, hydrolytic enzymes could not be demonstrated in these autophagosomes. The vacuole played a major role in the subsequent peroxisomal breakdown since it provided the enzymes required for proteolysis. Two basically similar mechanisms were observed with respect to the administration of vacuolar enzymes into the sequestered cell compartment. The first mechanism involved incorporation of a small vacuolar vesicle into the sequestered cell compartment. The delimiting membrane of this vacuolar vesicle subsequently disrupted, thereby exposing the contents of the sequestered cell compartment to vacuolar hydrolases which then degraded the peroxisomal proteins. The second mechanism, observed in cells which already contained one or more autophagic vacuoles, included fusion of the delimiting membranes of an autophagosome with the membrane surrounding an autophagic vacuole which led to migration of the peroxisome inside the latter organelle. Peroxisomes of methanolgrown H. polymorpha were degraded individually. In one cell 2 or 3 peroxisomes might be subject to degradation at the same time, but they were never observed together in one autophagosome. However, fusions of autophagic vacuoles in one cell were frequently observed. After inhibition of the cell's energy-metabolism by cyanide ions or during anaerobic incubations the formation of autophagosomes was prevented and degradation was not observed.
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  • 8
    ISSN: 1432-072X
    Keywords: Hansenula polymorpha ; Peroxisomes ; Methanol ; Dihydroxyacetone synthase ; Cell fractionation ; Immunocytochemistry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The subcellular localization of dihydroxyacetone synthase (DHAS) in the methylotrophic yeast Hansenula polymorpha was studied by various biochemical and immunocytochemical methods. After cell fractionation involving differential and sucrose gradient centrifugation of protoplast homogenates prepared from methanol-grown cells, DHAS cosedimented with the peroxisomal enzymes alcohol oxidase and catalase. Electron microscopy of this fraction showed that it contained mainly intact peroxisomes, whereas SDS-polyacrylamide gel electrophoresis revealed two major protein bands (75 and 78 kDa) which were identified as alcohol oxidase and DHAS, respectively. The localization of DHAS in peroxisomes was further established by immunocytochemistry. After immuno-gold staining carried out on ultrathin sections of methanol-grown H. polymorpha using DHAS-specific antibodies, labelling was confined to the peroxisomal matrix.
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  • 9
    ISSN: 1434-601X
    Keywords: 21.60.Ev ; 27.90.+b
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract TheK π=0− bands in even uranium nuclei were studied in the compound reactions231Pa(p, 2n)230U,230, 232Th(α,2n)232, 234U and236U(d, pn)236U. In-beamγ-rays were measured in coincidence with conversion-electrons, which were detected with an iron-free orange spectrometer. The negative-parity levels are observed up to intermediate spins (I〈13−). In addition, the 1− and 3− levels in230U were confirmed by a decay study with an isotope separated230Pa source. For the heavier isotopes (A≥232) the properties of theK π=0− bands (energies andγ-branchings) are consistent with a vibrational character of these bands. For230U theK π=0− band lies at rather low energy (E(1−)=367 keV), and the level spacings within this band are very similar to those of the isotones228Th and226Ra, which might indicate the onset of a stable octupole deformation.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 6 (1990), S. 87-97 
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; methylotrophic yeast ; microbodies ; peroxisome-deficient mutants ; alcohol oxidase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol-utilization-defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase.Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol- and ethanol utilizing-deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome-deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol.The mutations complemented and therefore were recessive mutations in different genes.
    Additional Material: 6 Ill.
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