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  • Saccharomyces cerevisiae
  • Springer  (77)
  • American Meteorological Society
  • Institute of Physics
  • 1995-1999  (77)
  • 1940-1944
  • 1997  (20)
  • 1996  (57)
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  • 1995-1999  (77)
  • 1940-1944
Year
  • 1
    ISSN: 1572-8773
    Keywords: EPR ; Saccharomyces cerevisiae ; uptake ; vanadate ; vanadyl
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO4 3- concentration and the growth was completely arrested at a concentration of 20 mM of VO4 3- in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A ‘mobile’ and an ‘immobile’ species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rotational correlation time τ r indicated the relative motional freedom at the macromolecular site. A strongly ‘immobilized’ vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO2+ ions with isolated cell walls.
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  • 2
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    Cellular and molecular life sciences 52 (1996), S. 1130-1135 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; mitochondria ; mRNA-specific translational activation ; synthetic genes ; gene regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mitochondrial gene expression in yeast,Saccharomyces cerevisiae, depends on translational activation of individual mRNAs by distinct proteins encoded in the nucleus. These nuclearly coded mRNA-specific translational activators are bound to the inner membrane and function to mediate the interaction between mRNAs and mitochondrial ribosomes. This complex system, found to date only in organelles, appears to be an adaptation for targeting the synthesis of mitochondrially coded integral membrane proteins to the membrane. In addition, mRNA-specific translational activation is a rate-limiting step used to modulate expression of at least one mitochondrial gene in response to environmental conditions. Direct study of mitochondrial gene regulation and the targeting of mitochondrially coded proteins in vivo will now be possible using synthetic genes inserted into mtDNA that encode soluble reporter/passenger proteins.
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  • 3
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    Cellular and molecular life sciences 52 (1996), S. 1033-1041 
    ISSN: 1420-9071
    Keywords: Ubiquitin ; yeast ; Saccharomyces cerevisiae ; Dictyostelium discoideum ; cytoskeleton ; mutants ; endocytosis ; actin ; myosin ; calmodulin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Endocytosis is a general term that is used to describe the internalization of external and plasma membrane molecules into the cell interior. In fact, several different mechanisms exist for the internalization step of this process. In this review we emphasize the work on the actin-dependent pathways, in particular in the yeastSaccharomyces cerevisiae, because several components of the molecular machinery are identified. In this yeast, the analysis of endocytosis in various mutants reveals a requirement for actin, calmodulin, a type I myosin, as well as a number of other proteins that affect actin dynamics. Some of these proteins have homology to proteins in animal cells that are believed to be involved in endocytosis. In addition, the demonstration that ubiquitination of some cell surface molecules is required for their efficient internalization is described. We compare the actin, myosin and ubiquitin requirements for endocytosis with recent results found studying these processes usingDictyostelium discoideum and animal cells.
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  • 4
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    Cellular and molecular life sciences 52 (1996), S. 1111-1116 
    ISSN: 1420-9071
    Keywords: Mitochondria ; mitochondrial inheritance ; cytoskeleton ; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; membrane proteins ; organelle movement ; mitochondrial morphology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mechanisms mediating the inheritance of mitochondria are poorly understood, but recent studies with the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe have begun to identify components that facilitate this essential process. These components have been identified through the analysis of conditional yeast mutants that display aberrant mitochondrial distribution at restrictive conditions. The analysis of these mutants has uncovered several novel proteins that are localized either to cytoskeletal structures or to the mitochondria themselves. Many mitochondrial inheritance mutants also show altered mitochondrial morphology and defects in maintenance of the mitochondrial genome. Although some inheritance components and mechanisms appear to function specifically in certain types of cells, other conserved proteins are likely to mediate mitochondrial behavior in all eukaryotic cells.
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  • 5
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; mitochondrial ribosomes ; peptidyl transferase ; Varl ribosomal protein ; gene relocation ; posttranscriptional rRNA modification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mitochondria posses their own ribosomes responsible for the synthesis of a small number of proteins encoded by the mitochondrial genome. In yeast,Saccharomyces cerevisiae, the two ribosomal RNAs and a single ribosomal protein, Varl, are products of mitochondrial genes, and the remaining approximately 80 ribosomal proteins are encoded in the nucleus. The mitochondrial translation system is dispensable in yeast, providing an excellent experimental model for the molecular genetic analysis of the fundamental properties of ribosomes in general as well as adaptations required for the specialized role of ribosomes in mitochondria. Recent studies of the peptidyl transferase center, one of the most highly conserved functional centers of the ribosome, and the Varl protein, an unusual yet essential protein in the small ribosomal subunit, have provided new insight into conserved and divergent features of the mitochondrial ribosome.
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  • 6
    ISSN: 1432-0983
    Keywords: Key words Omnipotent suppression ; Microtubules ; Respiratory deficiency ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  SUP35 and SUP45 genes determine the accuracy of translation at the stage of termination. We present indirect evidence indicating that these genes may also control some cellular process mediated by microtubules. A majority of sup35 and sup45 suppressor mutations confer supersensitivity to benomyl, the drug which de-polymerizes microtubules. In addition, data correlating phenotypic manifestations of sup45 suppressor mutations, involving sensitivity to benomyl, respiratory deficiency and a suppressor effect, are also presented.
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  • 7
    ISSN: 1432-0983
    Keywords: Key words D-ribulose-5-phosphate 3-epimerase ; D-ribose-5-phosphate ketol-isomerase ; Pentose-phosphate pathway ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have cloned and characterized the two remaining unknown genes of the non-oxidative part of the pentose-phosphate pathway of Saccharomyces cerevisiae encoding the enzymes D-ribulose-5-phosphate 3-epimerase (Rpe1p) and D-ribose-5-phosphate ketol-isomerase (Rki1p). Rpe1p has an unexpected high specific activity of 2148 mU × (mg protein)–1 in crude extracts. Deletion mutants of RPE1 show no enzyme activity and are unable to grow on D-xylulose. Unexpectedly, haploid rki1 deletion mutants are not viable. Functional expression of RKI1 was demonstrated following an increase of gene dosage in the haploid rki1 deletion mutant, which restored viability and specific D-ribose-5-phosphate ketol-isomerase activity. Both enzymes show high similarity to the deduced protein sequences of various open reading frames, expressed sequence tags or cDNAs from different organisms.
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  • 8
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    Current genetics 30 (1996), S. 461-468 
    ISSN: 1432-0983
    Keywords: Keywords DNA repair ; Methylation damage ; Epistasis analysis ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The major genotoxicity of methyl methanesulfonate (MMS) is due to the production of a lethal 3-methyladenine (3MeA) lesion. An alkylation-specific base-excision repair pathway in yeast is initiated by a Mag1 3MeA DNA glycosylase that removes the damaged base, followed by an Apn1 apurinic/ apyrimidinic endonuclease that cleaves the DNA strand at the abasic site for subsequent repair. MMS is also regarded as a radiomimetic agent, since a number of DNA radiation-repair mutants are also sensitive to MMS. To understand how these radiation-repair genes are involved in DNA methylation repair, we performed an epistatic analysis by combining yeast mag1 and apn1 mutations with mutations involved in each of the RAD3, RAD6 and RAD52 groups. We found that cells carrying rad6, rad18, rad50 and rad52 single mutations are far more sensitive to killing by MMS than the mag1 mutant, that double mutants were much more sensitive than either of the corresponding single mutants, and that the effects of the double mutants were either additive or synergistic, suggesting that post-replication and recombination-repair pathways recognize either the same lesions as MAG1 and APN1, or else some differ- ent lesions produced by MMS treatment. Lesions handled by recombination and post replication repair are not simply 3MeA, since over-expression of the MAG1 gene does not offset the loss of these pathways. Based on the above analyses, we discuss possible mechanisms for the repair of methylation damage by various pathways.
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  • 9
    ISSN: 1432-0983
    Keywords: Key words Transcriptional regulation ; Phospholipid biosynthesis ; Saccharomyces cerevisiae ; INO2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression of structural genes of phospholipid biosynthesis in yeast is mediated by the inositol/choline-responsive element (ICRE). ICRE-dependent gene activation, requiring the regulatory genes INO2 and INO4, is repressed in the presence of the phospholipid precursors inositol and choline. INO2 and, to a less extent, INO4 are positively autoregulated by functional ICRE sequences in the respective upstream regions. However, an INO2 allele devoid of its ICRE functionally complemented an ino2 mutation and completely restored inositol/choline regulation of Ino2p-dependent reporter genes. Low-level expression of INO2 and INO4 genes, each under control of the heterologous MET25 promoter, did not alter the regulatory pattern of target genes. Thus, upstream regions of INO2 and INO4 are not crucial for transcriptional control of ICRE-dependent genes by inositol and choline. Interestingly, over-expression of INO2, but not of INO4, counteracted repression by phospholipid precursors. Possibly, a functional antagonism between INO2 and a negative regulator is the key event responsible for repression or de-repression.
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  • 10
    ISSN: 1432-0983
    Keywords: Biocontrol ; Secretion ; Chitinase ; Expression cloning ; Saccharomyces cerevisiae ; Trichoderma harzianum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from aTrichoderma harzianum cDNA library by expression in yeast. The 1473-bpchil cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 fromBacillus circulans. TheT. harzianum endochitinase I was secreted into the culture medium by the yeastSaccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.
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  • 11
    ISSN: 1432-0983
    Keywords: Heat-shock response ; Multidrug resistance ; AP-1 homolog ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have examined whether the stress-induced transcriptional activation ofYDR1/PDR5/STS1 is mediated by yAP-1 and yAP-2. Of the stresses examined, heat shock-induced, rapid and transient PDR5 expression became very low in ayap1 yap2 double-gene disruptant, indicating that the yAP proteins mediate the response. Similar results were obtained withSNQ2, a close homologue ofPDR5. A set of 5′-truncation derivatives of thePDR5 gene identified the region from −484 to −434 as being sufficient for the response. A sequence similar to the yAP-1 recognition element recently identified in the stress-responsive yeast genes was found in this region and in the 5′-flanking sequences ofSNQ2.
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  • 12
    ISSN: 1432-0983
    Keywords: Key words Biocontrol ; Secretion ; Chitinase ; Expression cloning ; Saccharomyces cerevisiae ; Trichoderma harzianum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from a Trichoderma harzianum cDNA library by expression in yeast. The 1473-bp chi1 cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 from Bacillus circulans. The T. harzianum endochitinase I was secreted into the culture medium by the yeast Saccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.
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  • 13
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    Current genetics 31 (1997), S. 401-407 
    ISSN: 1432-0983
    Keywords: Key words Cytochrome oxidase ; Mitochondrial localization ; PET1402/OXA1 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast PET1402/OXA1 gene encoding a 44.8-kDa protein is required for mitochondrial biogenesis. Substitution of Leu240 to serine in the protein results in an accumulation of the precursor form of the mitochondrially encoded subunit 2 of cytochrome oxidase (Cox2) and temperature-sensitive respiration. This temperature sensitivity can be suppressed by a mutation in the cox2 gene changing Ala189 of the Cox2 protein to proline. In the cox2-ts1402 double mutant respiration is restored without removal of the Cox2 pre-sequence. The suppression suggests an interaction of the Pet1402 protein with the cytochrome oxidase complex. Antibodies raised against the predicted C-terminus and the tagged N-terminus of the Pet1402 protein reacted with a 37-kDa polypeptide. This protein, present in the mitochondrial fraction, is localized within the inner membrane. The difference in size can be explained by the removal of the predicted mitochondrial-targeting sequence from the Pet1402 protein. The mitochondrial localization of the protein points to a direct interaction with the cytochrome oxidase complex.
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  • 14
    ISSN: 1432-0983
    Keywords: Key words Cytochrome b ; Mutants ; Mitochondria ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nucleotide changes present in a group of five cytochrome b mit– mutants were analyzed at the sequence level. Two single-base changes were found: one (M10-152) generated a nonsense codon in the first exon while the other (M8-181) created a missense substitution in the second exon. The other mutants all have multiple (three) substitutions that either resulted in a missense mutation in a coding region (M17-162) or else changed nucleotides in the last intron of the gene, so blocking its excision (M6-200 and M8-53). The synthesis of mitochondrial polypeptides and the steady state concentration of the complex-III subunits were examined. The Rieske protein and the core-4 and core-5 subunits were much reduced in all mutants. Consequently the overall stability of complex III is very sensitive even to amino-acid substitutions in the cytochrome b protein. Mutant M8-53 provides direct evidence for the proposed role of the P9.1 stem in the core structure of the group-I type last intron of this gene.
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  • 15
    ISSN: 1432-0983
    Keywords: Key wordsβ-glucosidase ; Candida wickerhamii ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast Candida wickerhamii exports a cell-associated β-glucosidase that is active against cellobiose and all soluble cellodextrins. Because of its unique ability to tolerate end-product inhibition by glucose, the bglB gene that encodes this enzyme was previously cloned and sequenced in this laboratory. Using several different promoters and constructs, bglB was expressed in the hosts Escherichia coli, Pichia pastoris, and Saccharomyces cerevisiae. Expression was initially performed in E. coli using either the lacZ or tac promoter. This resulted in intracellular expression of the BglB protein with the protein being rapidly fragmented. Secretion and glycosylation of active β-glucosidase was achieved with several different S. cerevisiae constructs utilizing either the adh1 or the gal1 promoter on 2-µ replicating plasmids. When either the invertase (Suc2) or the BglB secretion signal was used, BglB protein remained associated with the cell wall and appeared to be hyperglycosylated. Expression in P. pastoris was also examined to determine if higher activity and expression could be achieved in a yeast host that usually does not hyperglycosylate. Using the alcohol oxidase promoter in conjunction with either the pho1 or the α-factor secretion signal, the recombinant enzyme was successfully secreted and glycosylated in P. pastoris. However, levels of protein expression from the chromosomally integrated vector were insufficient to detect activity.
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  • 16
    ISSN: 1432-0983
    Keywords: Key words Bleomycin hydrolase ; Saccharomyces cerevisiae ; Thiol proteases ; Protein amphitropism ; Processing of glycosyl-phosphatidylinositol (GPI) anchor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Bleomycin hydrolase, Blh1p, from yeast was co-purified with Gce1p, a cAMP-binding ectoprotein, anchored to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Blh1p is a hydrophilic thiol protease lacking transmembrane domains. We have used polyclonal antibodies to study the topology of the over-expressed protein in yeast and have found that it is amphitropic. Part of Blh1p is associated with plasma membranes, and most of the rest occurs in the cytosol. Both the growth conditions and calcium were found to have minor influences on the topology of Blh1p, in that glucose and the earth-alkali ion slightly enhanced recruitment to the membrane. We have examined the possibility that co-purification of Blh1p with Gce1p has a functional basis, and have observed that over-expression of BLH1 in yeast leads to an acceleration of the glucose-induced amphiphilic to hydrophilic conversion of Gce1p, wherein Blh1p could either directly catalyse the proteolytic removal of the polar headgroup of the GPI anchor subsequent to an initial lipolytic cleavage by a GPI-specific phospholipase C or indirectly modulate the reaction. The data show that a thiol protease is involved, but point to an indirect role of Blh1p in GPI processing. Proteases with similar or overlapping substrate specificity are likely to exist, since deletion of BLH1 neither entails a growth defect on any carbon source tested, nor the loss of proteolytic processing of the GPI anchor of Gce1p. Reduced proteolytic GPI processing is, however, observed in the blh1 mutant and the corresponding acceleration in the respective BLH1 multi-copy transformant.
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  • 17
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    Current genetics 29 (1996), S. 227-233 
    ISSN: 1432-0983
    Keywords: Trichoderma reesei ; β-Glucosidase ; Cellulase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An intronless form of thebgl1 gene encoding an extracellularβ-glucosidase fromTrichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL 1 promoter. Transformation of a yeast strain with this vector resulted in transformants that produce and secrete activeβ-glucosidase into the growth medium. Additionally, active recombinantβ-glucosidase protein was shown to be localized predominantly in the periplasmic space by using ap-nitrophenylβ-D-glycoside hydrolysis assay against fractionated yeast cells. The apparent size of the recombinant enzyme was 10–15 kDa larger than that of the native form. Treatment of the recombinantβ-glucosidase with endoglycosidase-H indicated the apparent increase in size was due to N-linked glycosylation.
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  • 18
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    Current genetics 29 (1996), S. 227-233 
    ISSN: 1432-0983
    Keywords: Key words  Trichoderma reesei ; β-Glucosidase ; Cellulase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract   An intronless form of the bgl1 gene encoding an extracellular β-glucosidase from Trichoderma reesei was expressed in the yeast Saccharomyces cerevisiae under the control of the yeast GAL1 promoter. Transformation of a yeast strain with this vector resulted in transformants that produce and secrete active β-glucosidase into the growth medium. Additionally, active recombinant β-glucosidase protein was shown to be localized predominantly in the periplasmic space by using a p-nitrophenyl β-D-glycoside hydrolysis assay against fractionated yeast cells. The apparent size of the recombinant enzyme was 10–15 kDa larger than that of the native form. Treatment of the recombinant β-glucosidase with endoglycosidase-H indicated the apparent increase in size was due to N-linked glycosylation.
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  • 19
    ISSN: 1432-0983
    Keywords: Key words Adenine biosynthesis ; ade8-18 ; ade2 mutations ; Red/white colony color assay ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the yeast Saccharomyces cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment, while epistatic mutations in the same pathway, i.e. ade8, preclude this phenomenon, resulting in normal white colonies. The shift in color from red to white (or vice versa) with a combination of appropriate wild-type and mutant alleles of the adenine-pathway genes has been widely utilized as a non-selective phenotype to visualise and quantify the occurrence of various genetic events such as recombination, conversion and aneuploidy. It has provided an invaluable tool for the study of gene dosage and plasmid stability. In competition experiments between disrupted ade2, ade8-18 transformants carrying either a functional or non-functional episomal ADE8 gene, we verified that white ade8 ade2 cells show a remarkable selective advantage over red ade2 cells, with important implications on the use of this assay for the monitoring of genetic events. The accumulation of the red pigment in ade2 cells is likely to be the cause for impaired growth in these cells.
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  • 20
    ISSN: 1432-0983
    Keywords: Key words RAD6 ; Ubiquitin-conjugating enzymes ; Saccharomyces cerevisiae ; Arabidopsis thaliana
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract AtUBC2 of Arabidopsis thaliana encodes a structural homolog of the RAD6 gene of Saccharomyces cerevisiae with approximately 65% identical amino acids. Like structural homologs from other organisms, AtUBC2 lacks the carboxyl-terminal extension of mostly acidic amino acids which is present in Rad6p. AtUBC2 was expressed in S. cerevisiae rad6 mutants. It was found to partially complement the UV sensitivity and reduced growth rate of rad6 mutants at elevated temperatures. AtUBC2 however, has no apparent influence on the degradation of N-end rule substrates in the heterologous host.
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  • 21
    ISSN: 1432-072X
    Keywords: Key words Plasma membrane H+-ATPase ; Saccharomyces cerevisiae ; Low pH ; PMA1 gene expression ; PMA2 gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cells of Saccharomyces cerevisiae grown in media with an initial pH of 2.5–6.0, acidified with a strong acid (HCl), exhibited the highest plasma membrane H+-ATPase-specific activity at an initial pH of 6.0. At a lower pH (above pH 2.5) ATPase activity (62–83% of the maximum level) still allowed optimal growth. At pH 2.5, ATPase activity was about 30% of the maximum value and growth was impaired. Quantitative immunoassays showed that the content of ATPase protein in the plasma membrane was similar across the entire pH range tested, although slightly lower at pH 2.5. The decrease of plasma membrane ATPase activity in cells grown at low pH was partially accounted for by its in vitro stability, which decreased sharply at pH below 5.5, although the reduction of activity was far below the values expected from in vitro measurements. Yeast growth under acid stress changed the pattern of gene expression observed at optimal pH. The level of mRNA from the essential plasma-membrane-ATPase-encoding gene PMA1 was reduced by 50% in cells grown at pH 2.5 as compared with cells grown at the optimal pH 5.0, although the content of ATPase in the plasma membrane was only modestly reduced. As observed in response to other kinds of stress, the PMA2 promoter at the optimal pH was up to eightfold more efficient in cells grown at pH 2.5, although it remained several hundred times less efficient than that of the PMA1 gene.
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  • 22
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    Journal of industrial microbiology and biotechnology 16 (1996), S. 102-109 
    ISSN: 1476-5535
    Keywords: Millet ; Pennisetum typhoides ; liquefaction ; saccharification ; baker's yeast ; Saccharomyces cerevisiae ; fermentation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of baker's yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4-α-d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca2+, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4-α-d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h−1, biomass yield coefficient of 0.5 g g−1 and feed substrate concentration of 200 g L−1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L−1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g−1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.
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  • 23
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    Molecular and cellular biochemistry 169 (1997), S. 95-106 
    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; N-glycosylation ; dolichol pathway ; ALG7 ; post-transcriptional regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The yeast ALG7 gene functions by initiating the synthesis of the dolichol-linked oligosaccharide precursor and plays an important role in the control of protein N-glycosylation. The levels of ALG7 multiple transcripts are modulated by the physiological status of the cell and environmental cues, and deregulation of their abundance is deleterious to several cellular functions. Since ALG7 mRNAs are unstable, we investigated the role of these transcripts' half-lives in determining their steady-state levels. Using a temperature-sensitive RNA polymerase II mutant, we demonstrate that increased stability was the primary determinant of higher ALG7 mRNA abundance in response to glucose limitation or treatment with tunicamycin. In contrast, at the G1/G0 transition point, changes in the decay rates were inversely related to ALG7 transcript accumulation: the decreased abundance of ALG7 mRNAs following exit from the mitotic cycle was associated with lengthening of the decay rates, while their increased accumulation after growth stimulation correlated with decreased stability. This suggests that, depending on the circumstance, mRNA half-lives can either directly determine the level of ALG7 transcript accumulation or oppose regulatory changes at other control levels.
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  • 24
    ISSN: 1573-4919
    Keywords: osmotic stress ; Saccharomyces cerevisiae ; glycerol ; K+/Na+ ions ; osmoregulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The intracellular level of Na+ and K+ of S. cerevisiae strain AB1375 revealed that under KCl as well as sorbitol stress, the cationic level was comparable to the level under no stress conditions. On the other hand, there was a sharp drop in the intracellular K+ content and increase in the Na+ content on addition of NaCl to the medium. However, the total cationic level was close to that under control conditions. In addition to changes in the cationic level, an enhanced production and accumulation of glycerol were also observed under osmotic stress. A regulatory mechanism co-ordinating the intracellular concentration of glycerol as well as Na+, K+ content under osmotic stress conditions has been proposed.
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  • 25
    ISSN: 1573-4943
    Keywords: Saccharomyces cerevisiae ; phosphoenolpyruvate carboxykinase ; pyridoxal phosphate ; site-directed mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V max/K mADP. These results indicate that Arg76 is not involved in CO2 binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207–210], does not perform an essential function for the enzyme activity.
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  • 26
    ISSN: 1573-5028
    Keywords: MAP kinase ; osmotic stress ; Pisum sativum ; Saccharomyces cerevisiae ; signal transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Previous analysis of the MAP kinase homologue from Pisum sativum (PsMAPK) revealed a potential MAP kinase motif homologous to that found in eukaryotic cdc2 kinases. Sequence comparison showed a 47% identity on amino acid sequence basis to the Saccharomyces cerevisiae Hog 1p MAP kinase involved in the osmoregulatory pathway. Under conditions of salt-stress aberrant morphology of a hog1 deletion mutant was completely restored and growth was partially restored by expression of the PsMAPK. This shows that PsMAPK is functionally active as a MAP kinase in S. cerevisiae. Comparison of PsMAPK with other kinases involved in osmosensitivity, showed a high degree of homology and implicates a possible role for PsMAPK in a P. sativum osmosensing signal transduction pathway.
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  • 27
    ISSN: 1617-4623
    Keywords: Key words Saccharomyces douglasii ; Saccharomyces cerevisiae ; CBP2 ; Mitochondria ; Pre-mRNA processing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  In Saccharomyces cerevisiae the only known role of the CBP2 gene is the excision of the fifth intron of the mitochondrial cyt b gene (bI5). We have cloned the CBP2 gene from Saccharomyces douglasii (a close relative of S. cerevisiae). A comparison of the S. douglasii and S. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that the S. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome, but not in the presence of an intronless S. cerevisiae mitochondrial genome. Also the S. douglasii and S. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from the S. douglasii mitochondrial genome.
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  • 28
    ISSN: 1617-4623
    Keywords: Pre-mRNA splicing ; Saccharomyces cerevisiae ; Suppressors ; prp24-1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The temperature-sensitiveprp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperaturesensitive (ts)prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic toprp21-1. This suppressor,prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that ofprp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in theprp24-1 strain. Genetic analysis of the suppressor showed thatprp21-2 is not a bypass suppressor ofprp24-1. The suppression ofprp24-1 byprp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2–U6 snRNA interactions.
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  • 29
    ISSN: 1617-4623
    Keywords: Saccharomyces douglasii ; Saccharomyces cerevisiae ; CBP2 ; Mitochondria ; Pre-mRNA processing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.
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  • 30
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Nucleotide excision repair ; RAD14 ; XPA homologue
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the accompanying paper we demonstrated that endonuclease III-sensitive sites in theMATα andHMLα loci ofSaccharomyces cerevisiae are repaired by the Nucleotide Excision Repair (NER) pathway. In the current report we investigated the repair of endonuclease III sites, 6-4 photoproducts and cyclobutane pyrimidine dimers (CPDs) in arad14-2 point mutant and in arad14 deletion mutant. TheRAD14 gene is the yeast homologue of the human gene that complements the defect in cells from xeroderma pigmentosum (XP) patients belonging to complementation group A. In the point mutant we observed normal repair of endonuclease III sites (i.e. as wild type), but no removal of CPDs at theMATα andHMLα loci. Similar experiments were undertaken using the recently createdrad14 deletion mutant. Here, neither endonuclease III sites nor CPDs were repaired inMAT a orHMR a. Thus the point mutant appears to produce a gene product that permits the repair of endonuclease III sites, but prevents the repair of CPDs. Previously it was found that, in the genome overall, repair of 6-4 photoproducts was less impaired than repair of CPDs in the point mutant. The deletion mutant repairs neither CPDs nor 6-4 photoproducts in the genome overall. This finding is consistent with the RAD14 protein being involved in lesion recognition in yeast. A logical interpretation is that therad14-2 point mutant produces a modified protein that enables the cell to repair endonuclease III sites and 6-4 photoproducts much more efficiently than CPDs. This modified protein may aid studies designed to elucidate the role of the RAD14 protein in lesion recognition.
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  • 31
    ISSN: 1617-4623
    Keywords: Key words RCC1 ; Saccharomyces cerevisiae ; Serine/threonine protein kinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The yeast PRP20 protein is homologous to the RCC1 protein of higher eukaryotes and is required for mRNA export and maintenance of nuclear structure. RCC1/PRP20 act as guanine nucleotide exchange factors for the nuclear Ras-like Ran/GSP1 proteins. In a search for prp20-10 allele-specific high-copy-number suppressors, the KSP1 locus, encoding a serine/threonine protein kinase was isolated. Ksp1p is a nuclear protein that is not essential for vegetative growth of yeast. Inactivation of the kinase activity by a mutation affecting the catalytic center of the Ksp1p eliminated the suppressing activity. Based on the isolation of a protein kinase as a high-copy-number suppressor, the phosphorylation of Prp20p was examined. In vivo labeling experiments showed that Prp20p is a phosphoprotein; however, deletion of the KSP1 kinase did not affect Prp20p phosphorylation.
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  • 32
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    Molecular genetics and genomics 253 (1996), S. 111-117 
    ISSN: 1617-4623
    Keywords: Key words MEL gene ; α-galactosidase ; Saccharomyces cerevisiae ; Saccharomyces paradoxus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  In order to study the molecular evolution of the yeasts grouped in the Saccharomyces sensu stricto species complex by analysis of the MEL gene family, we have cloned and sequenced two new species-specific MEL genes from Saccharomyces yeasts: S. paradoxus (MELp) and a Japanese Saccharomyces sp. (MELj). The clones were identified by sequence homology to the S. cerevisiae MEL1 gene. Both clones revealed an ORF of 1413 bp coding for a protein of 471 amino acids. The deduced molecular weights of the α-galactosidase enzymes were 52 767 for MELp and 52 378 for MELj. The nucleotide sequences of the MELp (EMBL accession no. X95505) and the MELj (EMBL accession no. X95506) genes showed 74.7% identity. The degree of identity of MELp to the MEL1 gene was 76.8% and to the S. pastorianus MELx gene, 75.7%. The MELj coding sequence was 75.1% identical to the MEL1 gene and 80.7% to the MELx gene. The data suggest that MEL1, MELj, MELp, and MELx genes are species-specific MEL genes. The strains studied each have only one MEL locus. The MELp gene is located on the S. paradoxus equivalent of S. cerevisiae chromosome X; the MELj gene was on the chromosome that comigrates with the S. cerevisiae chromosome VII/XV doublet and hybridizes to the S. cerevisiae chromosome XV marker HIS3.
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  • 33
    ISSN: 1617-4623
    Keywords: Key words DEAD-box protein ; DED1 ; RCC1 ; Saccharomyces cerevisiae ; SRM1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The Saccharomyces cerevisiae temperature-sensitive mutants srm1-1, mtr1-2 and prp20-1 carry alleles of a gene encoding a homolog of mammalian RCC1. In order to identify a protein interacting with RCC1, a series of suppressors of the srm1-1 mutation were isolated as cold-sensitive mutants and one of the mutants, designated ded1-21, was found to be defective in the DED1 gene. The double mutant, srm1-1 ded1-21, could grow at 35° C, but not at 37° C. A revertant of srm1-1 ded1-21 that became able to grow at 37° C acquired another mutation in the SRM1 gene, indicating the tight relationship between SRM1 and DED1. In all the rcc1 - strains examined, the amount of mutated SRM1 proteins was reduced or not detectable at the nonpermissive temperature. While mutated SRM1 protein was stabilized in all of the rcc1 - strains by the ded1-21 mutation, the ded1-21 mutation suppressed both srm1-1 and mtr1-2, but not the prp20-1 mutation, contrary to the previous finding that overproduction of the S. cerevisiae Ran homolog GSP1 suppresses prp20-1, but not srm1-1 or mtr1-2.
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  • 34
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    Molecular genetics and genomics 251 (1996), S. 682-691 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Two-hybrid system ; Protein-protein interactions ; PKC1 pathway ; MAP kinase cascade
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with theSaccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in thePKC1-mediated signalling pathway. Subsequently, Pkc1p and the otherPKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of thePKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.
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  • 35
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    Molecular genetics and genomics 251 (1996), S. 707-715 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Flocculation ; Transcriptional regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A flocculation gene was cloned from aSaccharomyces cerevisiae ATCC60715 genomic library, known to contain theFLO8 gene, on the basis of its ability to confer a flocculation phenotype on a non-flocculent strain. From a total of 11 130 clones, four clones sharing the several restriction fragments were isolated, suggesting that these were derived from the same locus. The results of integration mapping and disruption of the cloned gene indicated that this gene was theFLO8 gene. After disruption of theFLO8 gene, the strain lost its ability to flocculate. The DNA sequence of theFLO8 gene was determined. This gene includes a 2187-bp open reading frame that encodes a 729-amino acid protein. Computer analysis indicated that theFLO8 gene has a significant degree of homology with aS. cerevisiae chromosome V DNA sequence, but no homology with theFLO1 gene. The hydrophobicity profile of the putativeFLO8 gene product did not indicate the presence of any significantly hydrophobic regions. Southern analysis of theFLO8 gene present in various yeast strains indicated that theFLO8 gene is highly conserved in yeast strains having a variety of flocculation phenotypes and genotypes. Northern analysis revealed that the level ofFLO1 gene transcription is dependent on the rate of transcription of theFLO8 gene. These results suggest that theFLO8 gene mediates flocculation via transcriptional activation of theFLO1 gene.
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  • 36
    ISSN: 1617-4623
    Keywords: Key words Calcineurin ; Mpk1 MAP kinase ; Type II membrane protein ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Saccharomyces cerevisiae crv mutants (crv1, 2, 3 and 4) exhibit phenotypes, such as calcium resistance and vanadate sensitivity, which are apparently similar to those of calcineurin-deficient mutants. We have cloned and characterized the CRV4 gene that complements all the phenotypes of the crv4 mutant. DNA sequencing revealed that CRV4 is identical to the previously cloned gene TTP1, which encodes a type II membrane protein of unknown function. Deletion of CRV4/TTP1 causes no obvious phenotype except for Ca2+ resistance and vanadate sensitivity, but is synthetically lethal in combination with a deletion of MPK1, in a manner which is suppressible by the addition of an osmotic stabilizer. In medium containing sorbitol as an osmotic stabilizer, the cnb1 mpk1 ttp1 triple mutant exhibits a more severe growth defect than does any of the double mutants cnb1 ttp1, cnb1 mpk1 or mpk1 ttp1. A high Ca2+ concentration (50 mM) or a constitutively active form of calcineurin partially suppresses the growth defect of the mpk1 ttp1 double mutant. These results indicate that Ttp1 participates in a cellular event essential for growth and morphogenesis, in parallel with the pathways involving Mpk1 MAP kinase and calcineurin.
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  • 37
    ISSN: 1617-4623
    Keywords: Key words N-linked glycosylation ; Endoplasmic reticulum ; Oligosaccharyltransferase ; STT3 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract N-linked protein glycosylation is an essential process in eukaryotic cells. In the central reaction, the oligosaccharyltransferase (OTase) catalyzes the transfer of the oligosaccharide Glc3Man9GlcNAc2 from dolicholpyrophosphate onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum. The product of the essential gene STT3 is required for OTase activity in vivo, but is not present in highly purified OTase preparations. Using affinity purification of a tagged Stt3 protein, we now demonstrate that other components of the OTase complex, namely Ost1p, Wbp1p and Swp1p, specifically co-purify with the Stt3 protein. In addition, different conditional stt3 alleles can be suppressed by overexpression of either OST3 and OST4, which encode small components of the OTase complex. These genetic and biochemical data show that the highly conserved Stt3p is a component of the oligosaccharyltransferase complex.
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  • 38
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    Molecular genetics and genomics 250 (1996), S. 395-404 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; DNase ; Eukaryotes ; Genetic recombination
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Site-specific endonucleases have been found in various eukaryotic organelles such as mitochondria, chloroplasts and nuclei. These endonucleases initiate site-specific or homologous gene conversion in mitochondrial and nuclear DNA. Here, we report a new site-specific endonuclease activity, Endo.SK1, identified in mitochondria of strain SK1, a homothallic diploid strain ofSaccharomyces cerevisiae. Nucleotide sequences around the Endo.SK1-cleavage sites are different from those of known yeast site-specific endonucleases. The Endo.SK1 activity is, at least partly, specified by a gene in the SK1-derived mitochondria. A novel feature of the Endo.SK1 activity is its inducibility: the endonuclease activity was induced by ca. 40-fold by transfer of cells from a glucose medium into an acetate medium, and was then repressed. This transient induction was independent of the ploidy level of the cells, and coincided with induction of fumarase, a mitochondrial enzyme involved in the TCA cycle. Co-induction and co-repression of the mitochondrial site-specific endonuclease activity and a respiration-related enzyme indicate that the endonuclease activity is regulated in response to physiological conditions, and suggest a possible role for the endonuclease in mitochondrial DNA metabolism.
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  • 39
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Calcineurin ; MAP kinase cascade ; Pheromone-induced growth arrest ; Synthetic effect
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Saccharomyces cerevisiae mutants which exhibit phenotypes (calcium resistance and vanadate sensitivity) similar to those of calcineurin-deficient mutants were isolated. The mutants were classified into four complementation groups (crv1,2,3 and4).crv1 was allelic tocnb1, a mutation in the regulatory subunit of calcineurin. The nucleotide sequences ofCRV2 andCRV3 genes which complemented thecrv2 andcrv3 mutations, respectively, are identical to those ofBCK1/SLK1/SKC1/SSP31 andMPK1/SLT2, respectively, which are both involved in the MAP kinase cascade. A calcineurin-deletion mutation (Δcnb1), which by itself has no detectable effect on growth and morphology, enhanced some phenotypes (slow growth and morphological abnormality) ofcrv2 andcrv3 mutants. These phenotypes ofcrv2 andcrv3 mutants were partially suppressed by Ca2+ or by overproduction of the calcineurin subunits (Cmp2 and Cnb1). Like the calcineurin-deficient mutant,crv2 andcrv3 mutants were defective in recovery from α-factor-induced growth arrest. The defect in recovery of the Δcnb1 mutant was suppressed by overexpression ofMPK1. These results indicated that the calcineurin-mediated and the Mpk1- (Bck1-) mediated signaling pathways act in parallel to regulate functionally redundant cellular events important for growth.
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  • 40
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    Protoplasma 198 (1997), S. 177-185 
    ISSN: 1615-6102
    Keywords: Yeast ; Saccharomyces cerevisiae ; Mitochondrial nucleoids ; DNA-binding proteins ; Anaerobic culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The yeast cellsSaccharomyces cerevisiae grown up to stationary phase under either anaerobic conditions, or aerobic conditions in the presence of a respiratory inhibitor, antimycin A, had distinctive giant mitochondrial nucleoids (mt-nucleoids) (apparent diameter 0.6–0.9 μm) in contrast with the small mt-nucleoids (apparent diameter 0.2–0.4 μm) in respiratory-sufficient cells grown aerobically, as revealed by DAPI-fluorescence microscopy. The cytoplasmic respiratory-deficient cells (rho− cells), which were induced by treatment of wild-type cells with ethidium bromide, showed both giant and small mt-nucleoids of irregular size. In order to examine the structural and functional differences between giant and small mt-nucleoids, the former were successfully isolated from spheroplasts of three different cells by differential centrifugation and centrifugation on a discontinuous sucrose gradient. The isolated giant mt-nucleoids were intact in the morphology and were free of significant contamination by nuclear chromatin. The number of protein components involved in each of three different giant mt-nucleoids was similar to the number in small mt-nucleoids from aerobically grown cells, though a few noticeable differences were also recognized. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 26 kDa, and 20 kDa were the main components of small mt-nucleoids from aerobically grown cells as detected by chromatography on native DNA-cellulose. In contrast, the 67 kDa and 52 kDa proteins were hardly detected in corresponding fractions of giant mt-nucleoids from anaerobically grown cells and from rho− cells grown aerobically. On the other hand, mt-nucleoids from aerobically grown cells in the presence of antimycin A seemed to lack the 67 kDa protein but to have a small amount of the 52 kDa protein. This is the first demonstration of the variance of protein species involved in yeast mt-nucleoids according to the respiratory activity of mitochondria.
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  • 41
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Dynein ; Gene disruption ; cin8
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract Cytoplasmic dynein is a multisubunit, microtubule-dependent motor enzyme that has been proposed to function in a variety of intracellular movements. As part of an effort to understand the evolution and the biological roles of cytoplasmic dynein, we have identified the first non-metazoan dynein light chain 1, SLC1, in the yeastSaccharomyces cerevisiae. The amino acid sequence of the SLC1 protein is similar to those of the human,Drosophila andCaenorhabditis cytoplasmic dynein light chains 1. TheSLC1 gene lies adjacent to theYAP2 (CAD1) transcription unit. TheSLC1 coding sequence is split by two introns and its mRNA is detectable throughout the cell cycle. Tetrad analysis of heterozygotes harboring aTRP insertion in theSLC1 coding region indicate thatSLC1 function is not essential for cell viability. Furthermore, we demonstrate that double mutants, defective forSLC1 and the kinesin-relatedCIN8 genes are non-lethal. The redundancy ofSLC1 function in yeast contrasts with the cell death caused by loss-of-function mutations in the dynein light chain 1 gene inDrosophila melanogaster.
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  • 42
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    Molecular genetics and genomics 251 (1996), S. 153-160 
    ISSN: 1617-4623
    Keywords: Key words hUBC9 ; Saccharomyces cerevisiae ; Ubc9p ; Yeast centromere proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of the Saccharomyces cerevisiae centromere DNA-binding core complex, CBF3. The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity to S. cerevisiae Ubc9p and 64% identity to Schizosaccharomyces pombe (Sp) hus5. Overexpression of hUBC9 partially suppresses a S. cerevisiae ubc9 temperature-sensitive mutation, indicating that the UBC9 gene family is also functionally conserved. Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex. However, S. cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction between S. cerevisiae Ubc9 and Cbf3p proteins. The function of Ubc9p in the G2/M phase of S. cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis. Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation. We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro.
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  • 43
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Cell cycle ; Dox-A2 ; NIN1 ; P91A ; SUN2
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Abstract NIN1 is an essential gene for growth of the yeastSaccharomyces cerevisiae and was recently found to encode a component of the regulatory subunit of the 26S proteasome. Thenin1-1 mutant is temperature sensitive and its main defect is in G1/S progression and G2/M progression at non-permissive temperatures. One of the two multicopy suppressors ofnin1-1, SUN2 (SUppressor of Nin1-1), was found to encode a protein of 523 amino acids whose sequence is similar to those ofDrosophila melanogaster diphenol oxidase A2 and the mouse mast-cell Tum− transplantation antigen, P91A. The C-terminal half of Sun2p was found to be functional as Sun2p at 25° C, 30° C, and 34° C but not at 37° C. The open reading frame (ORF) of theDrosophila diphenol oxidase A2 gene (Dox-A2) was obtained from a lambda phage cDNA library using the polymerase chain reaction technique. TheDox-A2 ORF driven by theTDH3 promoter complemented the phenotype of a strain deleted forsun2. ThisDox-A2-dependent strain was temperature sensitive and accumulated dumb-bell-shaped cells, with an undivided nucleus at the isthmus, after temperature upshift. This morphology is similar to that ofnin1-1 cells kept at a restrictive temperature. These results suggest thatSUN2 is a functional counterpart ofDox-A2 and that these genes play a pivotal role in the cell cycle in each organism.
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  • 44
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Cell cycle ; LTE1 ; CDC15 ; CDC14
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract LTE1 encodes a homolog of GDP-GTP exchange factors for the Ras superfamily and is required at low temperatures for cell cycle progression at the stage of the termination of M phase inSaccharomyces cerevisiae. We isolated extragenic suppressors which suppress the cold sensitivity oflte1 cells and confer a temperature-sensitive phenotype on cells. Cells mutant for the suppressor alone were arrested at telophase at non-permissive temperatures and the terminal phenotype was almost identical to that oflte1 cells at non-permissive temperatures. Genetic analysis revealed that the suppressor is allelic toCDC15, which encodes a protein kinase. Thecdc15 mutations thus isolated were recessive with regard to the temperature-sensitive phenotype and were dominant with respect to suppression oflte1. We isolatedCDC14 as a low-copy-number suppressor ofcdc15-rlt1.CDC14 encodes a phosphotyrosine phosphatase (PTPase) and is essential for termination of M phase. An extra copy ofCDC14 suppressed the temperature sensitivity ofcdc15-rlt1 cells, but not that ofcdc15-1 cells. In addition, some residues that are essential for the Cdc14 PTPase activity were found to be non-essential for the suppression. These results strongly indicate that Cdc14 possesses dual functions; PTPase activity is needed for one function but not for the other. We postulate that the cooperative action of Cdc14 and Cdc15 plays an essential role in the termination of M phase.
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  • 45
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    Molecular genetics and genomics 251 (1996), S. 556-564 
    ISSN: 1617-4623
    Keywords: Key words cAMP ; Ca2+ ; Golgi apparatus ; Bud growth ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Biochemical and physiological studies have implicated cAMP and cAMP-dependent protein kinase (PKA) in a plethora of essential cellular processes. Here we show that yeast cells partially depleted of PKA activity (due to a tpk w mutation) and bearing a lesion in a Golgi-localized Ca2+ pump (Pmr1), arrest division with a small bud. The bud morphology of the arrested tpk1 w pmr1 mutant cells is characteristic of cells in S phase; however, the terminal phenotype of processes such as DNA replication and nuclear division suggests arrest at the G2/M boundary. This small bud, G2-arrest phenotype is similar to that of strains with a defect in cell wall biosynthesis (pkc1) or membrane biogenesis (och1); however, the biochemical defect may be different since the tpk1 w pmr1 double mutants retain viability. The growth defect of the tpk1 w pmr1 mutant can be alleviated by preventing the increase in cellular cAMP levels that is known to be associated with a decrease in PKA activity, or by supplementing the medium with millimolar amounts of Ca2+. Although the biochemical consequences of this increase in cAMP concentration are not known, the small-bud phenotype of the double mutant and the known protein processing defect of the pmr1 lesion suggest that the localization or function of some membrane component might be compromised and susceptible to perturbations in cellular cAMP levels. One candidate for such a protein is the cAMP-binding membrane ectoprotein recently described in yeast.
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  • 46
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    Molecular genetics and genomics 251 (1996), S. 556-564 
    ISSN: 1617-4623
    Keywords: cAMP ; Ca2+ ; Golgi apparatus ; Bud growth ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Biochemical and physiological studies have implicated cAMP and cAMP-dependent protein kinase (PKA) in a plethora of essential cellular processes. Here we show that yeast cells partially depleted of PKA activity (due to atpk w mutation) and bearing a lesion in a Golgi-localized Ca2+ pump (Pmr1), arrest division with a small bud. The bud morphology of the arrestedtpk1 w pmr1 mutant cells is characteristic of cells in S phase; however, the terminal phenotype of processes such as DNA replication and nuclear division suggests arrest at the G2/M boundary. This small bud, G2-arrest phenotype is similar to that of strains with a defect in cell wall biosynthesis (pkc1) or membrane biogenesis (och1); however, the biochemical defect may be different since thetpk1 w pmr1 double mutants retain viability. The growth defect of thetpk1 w pmr1 mutant can be alleviated by preventing the increase in cellular cAMP levels that is known to be associated with a decrease in PKA activity, or by supplementing the medium with millimolar amounts of Ca2+. Although the biochemical consequences of this increase in cAMP concentration are not known, the small-bud phenotype of the double mutant and the known protein processing defect of thepmr1 lesion suggest that the localization or function of some membrane component might be compromised and susceptible to perturbations in cellular cAMP levels. One candidate for such a protein is the cAMP-binding membrane ectoprotein recently described in yeast.
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  • 47
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    Molecular genetics and genomics 253 (1996), S. 173-181 
    ISSN: 1617-4623
    Keywords: Key words Illegitimate recombination ; Single-stranded plasmids ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome.
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  • 48
    ISSN: 1617-4623
    Keywords: Key words Rat ; DNA topoisomerase IIα ; Saccharomyces cerevisiae ; top2ts ; Complementation ; Leucine zipper
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  A series of yeast expression plasmids which comprise segments of the cDNA sequences encoding rat topo IIα have been constructed. The transcription of these constructs is under the control of the yeast GAL1 promoter. Galactose-dependent expression of the cloned rat topo IIα cDNA complemented a yeast top2 ts mutation, as well as a deletion mutation at the yeast TOP2 locus. Truncation of 12 N-terminal amino acids and/or 158 C-terminal amino acids of rat topo IIα had no effect on its ability functionally to substitute for top2 ts . Moreover, a cDNA construct with mutated putative leucine zipper domain (amino acids 993–1013) retained the complementation activity. These observations suggest that transformants capable of conditional topo IIα expression can be exploited as a useful model system for studies on the structure-function relationships of wild-type and mutated topo IIα, as well as the interplay of potential antitumor drugs with the enzyme.
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  • 49
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    Molecular genetics and genomics 253 (1996), S. 393-396 
    ISSN: 1617-4623
    Keywords: Key words Eukaryotic regulatory genes ; ADE2 gene ; Marked homologous recombination ; Gene-gene interference ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract  A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of high dosage of any given gene. It is based on the possibility of monitoring the presence in cells of a plasmid carrying the ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination in yeast. Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage. Possible implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general approaches to gene selection, such as the gene-gene interference method, are discussed.
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  • 50
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Chitin synthases ; Septum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Predicted protein sequences of fungal chitin synthases can be divided into a non-homologous N-terminal region and a C-terminal region that shows significant homology among the various synthases. We have explored the function of these domains by constructing a series of nested deletions, extending from either end, in theCHS1 andCHS2 genes ofSaccharomyces cerevisiae. In both cases, most or all of the sequences encoding the non-homologous N-terminal region (one-third of the protein for Chs1p and about one-fourth for Chs2p) could be excised, with little effect on the enzymatic activity in vitro of the corresponding synthase or on its function in vivo. However, further small deletions (20–25 amino acids) into the homologous region were deleterious to enzymatic activity and function, and often led to changes in the zymogenic character of the enzymes. Similarly, relatively small (about 75 amino acids) deletions from the C-terminus resulted in loss of enzymatic activity and function of both synthases. Thus, it appears that all the information necessary for membrane localization, enzymatic activity and function resides in the homologous regions of Chs1p and Chs2p, a situation that may also apply to other chitin synthases.
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  • 51
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Oxidative stress ; Pentose phosphate pathway ; Ribulose 5-phosphate epimerase
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    Topics: Biology
    Notes: Abstract We have isolated several mutants ofSaccharomyces cerevisiae that are sensitive to oxidative stress in a screen for elevated sensitivity to hydrogen peroxide. Two of the sixteen complementation groups obtained correspond to structural genes encoding enzymes of the pentose phosphate pathway. Allelism of thepos10 mutation (POS forperoxidesensitivity) to thezwf1/met1 mutants in the structural gene for glucose 6-phosphate dehydrogenase was reported previously. The second mutation,pos18, was complemented by transformation with a yeast genomic library. The open reading frame of the isolated gene encodes 238 amino acids. No detectable ribulose 5-phosphate epimerase activity was found in thepos18 mutant, suggesting that the corresponding structural gene is affected in this mutant. For that reason the gene was renamedRPE1 (forribulose 5-phosphateepimerase).RPE1 was localized to chromosome X. The predicted protein has a molecular mass of 25 966 Daltons, a codon adaptation index (CAI) of 0.32, and an isoelectric point of 5.82. Database searches revealed 32 to 37% identity with ribulose 5-phosphate epimerases ofEscherichia coli, Rhodospirillum rubrum, Alcaligenes eutrophus andSolanum tuberosum. We have characterizedRPE1 by testing enzyme activities inrpe1 deletion mutants and in strains that overexpressRPE1, and compared the hydrogen peroxide sensitivity ofrpe1 mutants to that of other mutants in the pentose phosphate pathway. Interestingly, all mutants tested (glucose 6-phosphate dehydrogenase, gluconate 6-phosphate dehydrogenase, ribulose 5-phosphate epimerase, transketolase, transaldolase) are sensitive to hydrogen peroxide.
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  • 52
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    Molecular genetics and genomics 255 (1997), S. 460-466 
    ISSN: 1617-4623
    Keywords: Key words Transcription ; TFIIH ; MAT1 ; RING finger protein ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Kin28/Ccl1, a cyclin-dependent kinase, is essential for the in vivo phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II in Saccharomyces cerevisiae. In a search for mutations co-lethal with a thermosensitive kin28 mutation, we have identified genes whose products interact functionally with Kin28. In the present work, we have studied a new complementation group of synthetic lethal mutations. The corresponding gene, RIG2, encodes a predicted RING finger protein. Rig2 is likely to be a homolog of MAT1 of higher eukaryotes which forms a ternary complex with MO15(cdk7) and cyclin H. Our genetic data suggest that Rig2 is a component of transcription factor TFIIH. Transcription activity in a rig2-ts mutant is impeded at restrictive temperature. However, none of the rig2-ts mutants obtained was UV sensitive, suggesting that Rig2 is dispensable for nucleotide excision repair.
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  • 53
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    Molecular genetics and genomics 256 (1997), S. 45-53 
    ISSN: 1617-4623
    Keywords: Key words Protein phosphorylation ; DNA replication ; Cell cycle checkpoint ; Saccharomyces cerevisiae
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    Notes: Abstract This article presents the identification and characterization of the PAK1 gene of Saccharomyces cerevisiae, and the biochemical characterization of the protein kinase activity that it encodes. Overexpression of the PAK1 gene product suppresses temperature-sensitive mutations of the pol1 (cdc17) gene, which encodes DNA polymerase α. Overexpression and suppression can be achieved either by expressing PAK1 from a high-copy-number plasmid, or by GAL1-induced transcription of PAK1. Gene disruption of PAK1 indicates that it is not an essential gene. The PAK1 gene encodes a protein with a kinase consensus domain. By deletion analysis and site-directed mutagenesis, we demonstrate that the complete and active kinase consensus domain is required for suppression. A glutathione-S-transferase (GST)-Pak1 fusion protein, overproduced in bacteria, can be purified in an active form with glutathione affinity beads or by immunoprecipitation. Thus, other protein subunits of Pak1 are not required for its activity. In vitro protein kinase assays show that GST-Pak1 can autophosphorylate, and can phosphorylate casein as an exogenous substrate. The phenotype of the suppressed cdc17-1 cells indicates that Pak1 suppression is inefficient and does not restore the wild-type phenotype. Pak1 suppression requires Rad9 function, but Pak1 does not affect Rad9 function. Overexpression of PAK1 does not enhance the expression of the POL1 gene. Pak1 may function by modifying and partially stabilizing thermolabile DNA polymerases, perhaps during DNA repair, because pak1 mutant cells are caffeine sensitive.
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  • 54
    ISSN: 1617-4623
    Keywords: DNA repair ; Nucleotide excision repair ; Saccharomyces cerevisiae
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    Notes: Abstract Ultraviolet irradiation of DNA induces cyclobutane pyrimidine dimers (CPDs), 6-4′-(pyrimidine 2′-one) pyrimidines and pyrimidine hydrates. The dimer is the major photoproduct, and is specifically recognized by endonuclease V of phage T4. Pyrimidine hydrates represent a small fraction of the total photoproducts, and are substrates for endonuclease III ofEscherichia coli. We used these enzymes to follow the fate of their substrates in the mating type loci ofSaccharomyces cerevisiae. In a RAD strain, CPDs in the transcriptionally activeMATα locus are preferentially repaired relative to the inactiveHMLα locus, whilst repair of endonuclease III-sensitive sites is not preferential. Therad1, 2, 3 and4 mutants, which lack factors that are essential for the incision step of nucleotide excision repair (NER), repair neither CPDs nor endonuclease III-sensitive sites, clearly showing that these lesions are repaired by the NER pathway. Previously it had been shown that the products of theRAD7 andRAD16 genes are required for the NER of CPDs from theHMLα locus. We show that, in the same locus, these gene products are not needed for removal of endonuclease III-sensitive sites by the same mechanism. This indicates that the components required for NER differ depending on either the type of lesion encountered or on the specific location of the lesion within the genome.
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  • 55
    ISSN: 1617-4623
    Keywords: RCC1 ; Saccharomyces cerevisiae ; Serine/threonine protein kinase
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    Notes: Abstract The yeast PRP20 protein is homologous to the RCC1 protein of higher eukaryotes and is required for mRNA export and maintenance of nuclear structure. RCC1/PRP20 act as guanine nucleotide exchange factors for the nuclear Ras-like Ran/GSP1 proteins. In a search forprp20-10 allele-specific high-copy-number suppressors, theKSP1 locus, encoding a serine/threonine protein kinase was isolated. Ksp1p is a nuclear protein that is not essential for vegetative growth of yeast. Inactivation of the kinase activity by a mutation affecting the catalytic center of the Ksp1p eliminated the suppressing activity. Based on the isolation of a protein kinase as a high-copy-number suppressor, the phosphorylation of Prp20p was examined. In vivo labeling experiments showed that Prp20p is a phosphoprotein; however, deletion of the KSP1 kinase did not affect Prp20p phosphorylation.
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  • 56
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; mRNA 3′ processing ; Poly(A) tail ; STS1 ; RNA15
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    Notes: Abstract In a search for proteins associated with Rna15p in processing the 3′ ends of messenger RNAs, we have looked for suppressors that correct, even partially, the thermosensitive growth defect of therna15-2 mutant. Mutations in a single locus that we namedSSM5, were able to suppress both the thermosensitivity of cell growth and the mRNA 3′ processing defect associated with therna15-2 mutation, but only slightly alleviated the thermosensitive growth defect of anrna14-1 mutant. Thessm5-1 mutant is sensitive to hydroxyurea at 37° C, a drug that inhibits DNA synthesis. By screening for complementation of the hydroxyurea-sensitive phenotype we cloned the corresponding wild-type gene and found that it corresponds to the essential geneSTS1 (also namedDBF8). Sts1p has an apparent molecular weight of 30 kDa and was confirmed to be a cytosolic protein by immunofluorescence analysis. Western blot analysis indicates that the thermosensitive mutant strainsrna15-2, rna14-1 andpap1-1 present a very low level of the Rna15p at 37° C. Thessm5-1 mutation restores the level of Rna15p in therna15-2 ssm5-1 double mutant. Use of the two-hybrid system suggests that Sts1p does not interact directly with Rna15p, but may be active as a homodimer. The present data suggest that Sts1p may play a role in the transport of Rna15p from the cytoplasm to the nucleus.
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  • 57
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    Molecular genetics and genomics 256 (1997), S. 469-480 
    ISSN: 1617-4623
    Keywords: Key words GMP kinase ; GDP-mannose synthesis ; N-glycosylation ; Mannose outer chain elongation ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract We have found that yeast mutants that are defective in mannose outer chain elongation of N-linked glycoproteins show higher cell wall porosity than normal cells, and are hypersensitive to antibiotics with a large molecular weight; such as neomycin and geneticin. Wild-type yeast cells also showed enhanced sensitivity to neomycin in the presence of tunicamycin, an inhibitor of N-glycosylation, suggesting that the extent of N-glycosylation may affect the sensitivity of yeast cells to drugs and that sensitivity to neomycin may be an effective method for screening for yeast mutants defective in N-glycosylation. Pursuing this logic, we isolated neomycin-sensitive yeast mutants and screened them for defects in N-glycosylation. The neomycin-sensitive, N-glycosylation-defective mutants fell into 15 complementation groups including alleles of the previously isolated temperature-sensitive nes mutants nes10, nes17, and nes25. Gene cloning revealed that NES10 was identical to SEC20, which is involved in ER-Golgi protein transport. NES17 was identical to ALG1, which encodes a β-1,4-mannosyltransferase present in the ER. MSN17, a multicopy suppressor of nes17/alg1, was also isolated and found to be an allele of PSA1, which is involved in GDP-mannose synthesis. NES25 was identical to GUK1, which encodes a GMP kinase. Overexpression of MSN17 increased the GDP-mannose level in a wild-type strain by about threefold, and guk1 decreased the GDP-mannose level to one-fourth, suggesting a close relationship between GTP metabolism and mannose outer chain elongation; the link is presumably provided by the process of GDP-mannose transport in the Golgi membranes.
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  • 58
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Mitochondrial pre-mRNA splicing ; NAM2 gene ; Leucyl tRNA synthetase ; RNA maturase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract TheNAM2 gene ofSaccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrialcytb gene (bI4) and the fourth intron of the mitochondrialcoxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.
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  • 59
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Mitochondria ; Integral membrane protein
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    Notes: Abstract SSM4 was isolated as a suppressor ofrna14-1, a mutant involved in nuclear mRNA maturation. In order to isolate genes interacting withSSM4, we have searched for mutants that are syntheticlethal in association with anSSM4 deletion. Among the mutants obtained, one, namedsls1-1, shows apet − phenotype. We have cloned and sequenced this gene. It encodes a protein with a calculated molecular mass of 73 kDa. This protein contains a mitochondrial targeting presequence but does not show homology with other known proteins. Deletion ofSLS1 does not affect cell viability on glucose but is lethal on a non-fermentable medium. The Sls1p protein does not appear to be involved in mitochondrial DNA replication, transcription, or in RNA splicing maturation or stability. We have also tagged this protein and localized it in mitochondria. Treatment with alkaline carbonate does not extract this protein from mitochondria, suggesting strongly that it is a mitochondrial integral membrane protein. Thus, theSLS1 gene, encodes a mitochondrial integral membrane protein and is paradoxically synlethal in association with a deletion of theSSM4 gene, which encodes an integral nuclear membrane protein.
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  • 60
    ISSN: 1617-4623
    Keywords: Key words DNA replication ; Meiosis ; Saccharomyces cerevisiae
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    Notes: Abstract  In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae. DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase α-primase complex. Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared. We show that premeiotic DNA synthesis does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. At late meiotic times, ime2Δ diploids exhibit an additional round of DNA synthesis. Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase α is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle. Moreover, the catalytic subunit and the B-subunit of DNA polymerase α are specifically degraded during spore formation. Phosphorylation of the B-subunit does not occur in ime1Δ diploids, but does occur in ime2Δ diploids with an 8–9 h delay. In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation.
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  • 61
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    Molecular genetics and genomics 256 (1997), S. 682-689 
    ISSN: 1617-4623
    Keywords: Key words General regulatory factors ; Gal4 protein ; Saccharomyces cerevisiae ; Basal transcription
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    Topics: Biology
    Notes: Abstract Expression of the gene GCY1 in Saccharomycescerevisiae is induced by about 25-fold in the presence of galactose as a result of activation by Gal4p. In contrast to other Gal4p-regulated genes, such as GAL1 or GAL10, GCY1 is transcribed at a relatively high basal level. We have analysed the basis of this behaviour and have found that, in addition to a UAS GAL , a binding site for the general regulatory factor Reb1p is localized 100 bp upstream of the TATA sequence and about 140 bp 3′ to the UAS GAL . Reb1p binds to this site with low affinity. Reb1p, an abundant, multifunctional DNA-binding protein in yeast, acts as a weak transcriptional activator in the control regions of several genes encoding unrelated functions. The action of Reb1p is assumed to be strongly position dependent. In the control region of GCY1, Reb1p acts independently of position and stimulates basal expression of GCY1 about threefold, whereas Gal4p-mediated activation is not influenced significantly. Promoter-proximal insertion of an additional Reb1p recognition site enhances basal transcription only marginally, but can largely compensate for deletion of the natural Reb1p-binding site. Either an Abf1p- or a Rap1p-binding site can substitute for the Reb1p recognition sequence, indicating that these general regulatory factors fulfill related functions in basal transcription, without affecting Gal4p-mediated activation. In addition to Reb1p, the sequence of the Gal4p-binding site influences basal transcription. This effect is independent of the Gal4 protein, as it operates in a gal4 mutant background as well. This finding suggests that the nucleotide sequence of the UAS GAL in the GCY1 promoter has intrinsic properties, presumably a particular DNA structure, that influence basal transcription and act synergistically with Reb1p.
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  • 62
    ISSN: 1617-4623
    Keywords: Key words YAP1 ; Kluyveromyces lactis ; Saccharomyces cerevisiae ; Transcriptional activator ; Oxidative stress response
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    Topics: Biology
    Notes: Abstract The KlYAP1 gene, encoding the transcription factor Yap1p from Kluyveromyces lactis, was cloned by functional complementation of the cadmium hypersensitivity phenotype of a Saccharomyces cerevisiae strain lacking functional YAP1 and YAP2 genes. The KlYAP1 gene product is 41% identical to Yap1p, the sequence similarity being centered on the bZip domain and extending into the C-terminal portion of both proteins. When expressed in S. cerevisiae, this gene efficiently complements some of the phenotypes associated with both yap1 and yap2 mutations and also mediates AP-1 response element-dependent transcriptional activation in response to H2O2. Gene disruption experiments in K. lactis indicated that the KlYAP1 gene is involved in both the oxidative and cadmium response pathways. We also demonstrate the existence in K. lactis of inducible protective stress responses to both peroxides and superoxides and investigate the role of the Klyap1p protein in these responses.
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  • 63
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    Molecular genetics and genomics 250 (1996), S. 162-168 
    ISSN: 1617-4623
    Keywords: Key words DNA repair ; Regulation ; Gene fusion ; DRE element ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The interstrand cross-link repair gene SNM1 of Saccharomyces cerevisiae was examined for regulation in response to DNA-damaging agents. Induction of SNM1-lacZ fusions was detected in response to nitrogen mustard, cis-platinum (II) diamine dichloride, UV light, and 8-methoxypsoralen +UVA, but not after heat-shock treatment or incubation with 2-dimethylaminoethylchloride, methylmethane sulfonate or 4-nitroquinoline-N-oxide. The promoter of SNM1 contains a 15 bp motif, which shows homology to the DRE2 box of the RAD2 promoter. Similar motifs have been found in promoter regions of other damage-inducible DNA repair genes. Deletion of this motif results in loss of inducibility of SNM1. Also, a putative negative upstream regulation sequence was found to be responsible for repression of constitutive transcription of SNM1. Surprisingly, no inducibility of SNM1 was found after treatment with DNA-damaging agents in strains without an intact DUN1 gene, while regulation seems unchanged in sad1 mutants.
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  • 64
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    Molecular genetics and genomics 250 (1996), S. 162-168 
    ISSN: 1617-4623
    Keywords: DNA repair ; Regulation ; Gene fusion ; DRE element ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The interstrand cross-link repair geneSNM1 ofSaccharomyces cerevisiae was examined for regulation in response to DNA-damaging agents. Induction ofSNM1-lacZ fusions was detected in response to nitrogen mustard, cis-platinum (II) diamine dichloride, UV light, and 8-methoxypsoralen + UVA, but not after heat-shock treatment or incubation with 2-dimethyl-aminoethylchloride, methylmethane sulfonate or 4-nitroquinoline-N-oxide. The promoter ofSNM1 contains a 15 bp motif, which shows homology to the DRE2 box of theRAD2 promoter. Similar motifs have been found in promoter regions of other damage-inducible DNA repair genes. Deletion of this motif results in loss of inducibility ofSNM1. Also, a putative negative up-stream regulation sequence was found to be responsible for repression of constitutive transcription ofSNM1. Surprisingly, no inducibility ofSNM1 was found after treatment with DNA-damaging agents in strains without an intactDUN1 gene, while regulation seems unchanged insad1 mutants.
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  • 65
    ISSN: 1617-4623
    Keywords: Key words DNA repair ; Nucleotide excision repair ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Ultraviolet irradiation of DNA induces cyclobutane pyrimidine dimers (CPDs), 6-4′-(pyrimidine 2′-one) pyrimidines and pyrimidine hydrates. The dimer is the major photoproduct, and is specifically recognized by endonuclease V of phage T4. Pyrimidine hydrates represent a small fraction of the total photoproducts, and are substrates for endonuclease III of Escherichia coli. We used these enzymes to follow the fate of their substrates in the mating type loci of Saccharomyces cerevisiae. In a RAD strain, CPDs in the transcriptionally active MATα locus are preferentially repaired relative to the inactive HMLα locus, whilst repair of endonuclease III-sensitive sites is not preferential. The rad1, 2, 3 and 4 mutants, which lack factors that are essential for the incision step of nucleotide excision repair (NER), repair neither CPDs nor endonuclease III-sensitive sites, clearly showing that these lesions are repaired by the NER pathway. Previously it had been shown that the products of the RAD7 and RAD16 genes are required for the NER of CPDs from the HMLα locus. We show that, in the same locus, these gene products are not needed for removal of endonuclease III-sensitive sites by the same mechanism. This indicates that the components required for NER differ depending on either the type of lesion encountered or on the specific location of the lesion within the genome.
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  • 66
    ISSN: 1433-4909
    Keywords: Key words Hydrostatic pressure ; Saccharomyces cerevisiae ; Transient vacuolar acidification ; Vacuolar H+-ATPase ; Chemical reaction of CO2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We analyzed the vacuolar acidification in response to elevated hydrostatic pressure in Saccharomyces cerevisiae. The vacuolar pH, defined using 6-carboxyfluorescein, was directly measured in a hyperbaric chamber with a transparent window under high hydrostatic pressure. The vacuole of strain X2180 became acidified at the onset of pressurization to an extent dependent on the magnitude of pressure applied. A pressure of 40–60 MPa transiently reduced the vacuolar pH by about 0.33 within 4 min. The transient acidification was observed in the presence of D-glucose, D-fructose, or D-mannose as a carbon source, but not 3-o-methyl-D-glucose, ethanol, or glycerol, suggesting that the generation of CO2 was involved in the process. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH when hydrostatic pressure was applied. This result indicates that the transient vacuolar acidification induced by elevated hydrostatic pressure is mediated through the function of the vacuolar H+-ATPase.
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  • 67
    ISSN: 1573-5028
    Keywords: Brassica napus ; complementation ; DNA polymerase δ ; DNA replication ; proliferating cell nuclear antigen (PCNA) ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA encoding the proliferating cell nuclear antigen (PCNA) from Brassica napus (oilseed rape) was shown to complement the lethal deletion mutation in the PCNA gene (ΔPOL30) of Saccharomyces cerevisiae. We provide unequivocal evidence that the B. napus PCNA can perform all the essential functions of the yeast PCNA in DNA replication, although some species-specific differences may exist. In addition, the B. napus PCNA expressed as a fusion polypeptide with glutathione S-transferase (GST) was shown to stimulate the activity and processivity of two δ-like DNA polymerases from wheat in vitro. These experiments provide direct biochemical evidence that the B. napus PCNA may function as an auxiliary factor in plant cell DNA replication.
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  • 68
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    Journal of industrial microbiology and biotechnology 16 (1996), S. 117-123 
    ISSN: 1476-5535
    Keywords: baker's yeast ; Saccharomyces cerevisiae ; fed-batch cultivation ; ethanol sensor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Growth kinetics ofSaccharomyces cerevisiae in glucose syrup from cassava starch and sugarcane molasses were studied using batch and fed-batch cultivation. The optimum temperature and pH required for growth were 30°C and pH 5.5, respectively. In batch culture the productivity and overall cell yield were 0.31 g L−1 h−1 and 0.23 g cells g−1 sugar, respectively, on glucose syrup and 0.22 g L−1 h−1 and 0.18 g cells g−1 sugar, respectively, on molasses. In fed-batch cultivation, a productivity of 3.12 g L−1 h−1 and an overall cell yield of 0.52 g cells g−1 sugar in glucose syrup cultivation and a productivity of 2.33 g L−1 h−1 and an overall cell yield of 0.46 g cells g−1 sugar were achieved in molasses cultivation by controlling the reducing sugar concentration at its optimum level obtained from the fermentation model. By using an on-line ethanol sensor combined with a porous Teflon® tubing method in automating the feeding of substrate in the fed-batch culture, a productivity of 2.15 g L−1 h−1 with a yield of 0.47 g cells g−1 sugar was achieved using glucose syrup as substrate when ethanol concentration was kept at a constant level by automatic control.
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  • 69
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    Journal of industrial microbiology and biotechnology 17 (1996), S. 124-127 
    ISSN: 1476-5535
    Keywords: yeasts ; killer toxin ; fingerprinting ; Saccharomyces cerevisiae ; selected starters ; wine-making
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The extreme variability of the killer phenomenon in nature, expressed differently in different strains of the same yeast species, embodies an exceptional potential for the discrimination of yeasts at the strain level. Killer-sensitive relationships between a killer reference panel of 24 yeasts belonging to 13 species of six genera, and different industrial wine-starters ofSaccharomyces cerevisiae can be used profitably for a rapid and simple fingerprinting procedure.
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  • 70
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    Journal of industrial microbiology and biotechnology 17 (1996), S. 303-313 
    ISSN: 1476-5535
    Keywords: grape(s) ; wine yeast(s) ; Saccharomyces cerevisiae ; genetic analysis ; electrophoretic karyotyping ; segregation of chromosomal length polymorphism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Numerous studies have described the yeast biota of grapes, and grape must in order to understand better the succession of yeasts during fermentation of wine. The origin of the wine yeasts has been rather controversial. By using more elaborate isolation methods, classical genetic analysis and electrophoretic karyotyping of monosporic clones, with this study, credible proof now exists that the vineyard is the primary source for the wine yeasts and that strains found on the grapes can be followed through the fermentation process.
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  • 71
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    World journal of microbiology and biotechnology 14 (1997), S. 107-111 
    ISSN: 1573-0972
    Keywords: Glutaraldehyde ; immobilization ; monochloroacetic acid ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 72
    ISSN: 1572-9699
    Keywords: Sporulation ; Saccharomyces cerevisiae ; 2 deoxy glucose ; metabolic fluxes ; gluconeogenesis ; glyoxylate cycle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Quantitative studies of metabolic fluxes during Saccharomyces cerevisiae sporulation on acetate in the presence of the glucose analog, 2-deoxy glucose (2dG) are reported. We have studied the inhibition of sporulation and associated catabolic or anabolic fluxes by 2dG. Sporulation frequencies decreased from 50% to 2% asci per cell at 2dG concentrations in the range of 0.03 to 0.30 g l〉-1, respectively. Under the same conditions, the acetate consumption flux was inhibited up to 60% and the glyoxylate cycle and gluconeogenic fluxes decreased from 0.7 and 0.3 mmol h〉-1 g〉-1 dw, respectively, to negligible values. We observed a linear correlation of the acetate consumption rate with the sporulation frequency by varying the 2dG concentration. The linear correlation was also verified between the frequency of sporulation and the fluxes through glyoxylate cycle and gluconeogenic pathways. In addition, the same association of inhibition of sporulation and metabolic fluxes was found in other S. cerevisiae strains displaying different potentials of sporulation. The results presented suggest that inhibition of sporulation in the presence of the glucose analog may be attributed, at least in part, to the inhibition of anabolic fluxes and might be associated with catabolite repression.
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  • 73
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    Antonie van Leeuwenhoek 69 (1996), S. 257-265 
    ISSN: 1572-9699
    Keywords: acetate and oxygen consumption ; Saccharomyces cerevisiae ; sporulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have quantified yeast carbon and oxygen consumption fluxes and estimated anabolic fluxes through glyoxylate and gluconeogenic pathways under various conditions of sporulation on acetate. The percentage of sporulation reached a maximum of 55% to 60% after 48 h in sporulation medium, for cells harvested from logarithmic growth in acetate minimal medium. When cells were harvested in the stationary phase of growth before transfer to sporulation medium, the maximum percentage of sporulation decreased to 40% along with the occurrence of meiosis as could be judged by counting of bi- and tetra-nucleated cells. In both experiments, the rates of acetate and oxygen consumption decreased as a function of time when exposed to sporulation medium. Apparently, the decrease of metabolic rates was not due to alkalinization. By systematically varying the cell concentration in sporulation medium from 1.4×107 to 20×107 cell ml-1, the percentage of sporulating cells was found to decrease in parallel with the rate of acetate consumption. When the sporulation efficiency attained under the different experimental conditions was plotted as a function of the rate of acetate consumption, a linear correlation was found. Anabolic fluxes estimation revealed a decrease of the rate through gluconeogenic and glyoxylate pathways occurring during sporulation progression. The pattern of metabolic fluxes progressively evolved toward a predominance of more oxidative catabolic fluxes than those exhibited under growth conditions. The results obtained are discussed in terms of a characteristic pattern of metabolic fluxes and energetics, associated to the development of yeast sporulation.
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  • 74
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    Biodegradation 7 (1996), S. 277-286 
    ISSN: 1572-9729
    Keywords: Saccharomyces cerevisiae ; bioaccumulation ; chromium ; ground water
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Energy, Environment Protection, Nuclear Power Engineering , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Chromium can be removed from ground water by the unicellular yeast, Saccharomyces cerevisiae. Local ground water maintains chromium as CrO4 2- because of bicarbonate buffering and pH and E h conditions (8.2 and +343 mV, respectively). In laboratory studies, we used commercially available, nonpathogenic S. cerevisiae to remove hexavalent chromium [Cr(VI)] from ground water. The influence of parameters such as temperature, pH, and glucose concentration on Cr(VI) removal by yeast were also examined. S. cerevisiae removed Cr(VI) under aerobic and anaerobic conditions, with a slightly greater rate occurring under anaerobic conditions. Our kinetic studies reveal a reaction rate (Vmax) of 0.227 mg h-1 (g dry wt biomass)-1 and a Michaelis constant (Km) of 145 mg/l in natural ground water using mature S. cerevisiae cultures. We found a rapid (within 2 minutes) initial removal of Cr(VI) with freshly hydrated cells [55–67 mg h-1 (g dry wt biomass)-1] followed by a much slower uptake [0.6–1.1 mg h-1 (g dry wt biomass)-1] that diminished with time. A materials-balance for a batch reactor over 24 hours resulted in an overall shift in redox potential from +321 to +90 mV, an increase in the bicarbonate concentration (150–3400 mg/l) and a decrease in the Cr(VI) concentration in the effluent (1.9-0 mg/l).
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  • 75
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    World journal of microbiology and biotechnology 14 (1997), S. 243-246 
    ISSN: 1573-0972
    Keywords: Amino acid analogue ; Saccharomyces cerevisiae ; secondary products ; wine yeast ; winemaking
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Mutants resistant to the amino acid analogues dl-thiaisoleucine, dl-4-azaleucine, 5,5,5-trifluoro-dl-leucine and l-O-methylthreonine, were isolated from Saccharomyces cerevisiae wine yeast strains. The fermentative production of secondary metabolites by the mutants was tested in grape must. Higher alcohols, acetaldehyde and acetic acid concentration varied depending on strain and analogue. Most of the mutants produced increased amounts of amyl alcohol. A remarkable variability in the level of n-propanol, isobutanol, acetaldehyde and acetic acid was observed. In practical application, the use of mutants resistant to amino acid analogues can improve the quality of wines by reducing or increasing the presence of some secondary compounds.
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  • 76
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    World journal of microbiology and biotechnology 12 (1996), S. 25-27 
    ISSN: 1573-0972
    Keywords: Batch fermentation ; immobilization ; Saccharomyces cerevisiae ; secondary products ; wine yeast ; wine making
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Five, highly flocculeng strains of Saccharomyces cerevisiae, isolated from wine, were immobilized in calcium alginate beads to optimize primary must fermentation. Three cell-recycle batch fermentations (CRBF) of grape musts were performed with the biocatalyst and the results compared with those obtained with free cells. During the CRBF process, the entrapped strains showed some variability in the formation of secondary products of fermentation, particularly acetic acid and acetaldehyde. Recycling beads of immobilized flocculent cells is a good approach in the development and application of the CRBF system in the wine industry.
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  • 77
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    World journal of microbiology and biotechnology 13 (1997), S. 711-712 
    ISSN: 1573-0972
    Keywords: Endopolygalacturonase ; pectic enzymes ; Saccharomyces cerevisiae ; yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The laboratory strain of S. cerevisiae, IM1-8b, showed pectolytic activity in the presence of either glucose, fructose, or sucrose as the carbon source, but not with galactose. The enzyme activity was rapidly lost with shaking. The optimum pH and temperature for activity were 4.5 and 45°C, respectively. The enzyme was an endopolygalacturonase, since it preferentially hydrolysed pectate over pectin and decreased the viscosity of a 5% polygalacturonic solution by about 30% in 30min producing oligogalacturonic acid and digalacturonic acid as end-products.
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