bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polyploid fragile strains of Saccharomyces cerevisiae—a novel source of proteins for nutritional purposes
Authors: Stateva L. I. , Venkov P. V. , Hadjiolov A. A. , Koleva L. A. , Lyudskahov N. L. 
Published: May 2005

AbstractA series of prototrophic fragile strains of different ploidy (2n, 3n and 4n) has been genetically constructed on the basis ofhalopoid srb1 containing segregants of the fragile Saccharomyces cerevisiae mutant VY 1160. The strains have been characterized by several criteria. In regard to generation time, biomass yield, and nucleic acids content of the cells, the tetraploid srb1 homozygous hybrid is indistinguishable from an industrial strain of S. cerevisiae. However, it is characterized by a higher protein content. Unlikely any other laboratory or industrial strains, the original mutant and these hybrids possess an ability for lysis upon suspension in hypotonic solutions. The dependence of the percentge of lysed cells on the growth phase and concentration of osmotic stabilizer in the medium has been investigated. The quantity of proteins in the soluble fractions obtained after lysis of these strains by osmotic shock has been determined. These hybrids can be considered as a potential industrial source of potentials for nutritional purposes.
Membrane transport in an osmotically fragile mutant of Saccharomyces cerevisiae
Authors: Kotyk A. , Venkov P. , Dvor̆áková M. 
Published: May 2005

AbstractTransport properties of the osomotically fragile strain VY1160 of saccharomyces cerevisiae were compared with those of the parent S288c strain. Mediated diffusion of 6‐deoxy‐D‐glucose was practically unaffected; membrane‐potential dependent transport of D‐glucosamine was very much depressed in the fragile strain. The H+ ‐driven transport of L‐lysine and Lproline, as well as that of the hitherto uninvestigated D‐glucose‐6‐phosphate, were also very depressed. 2‐Deoxy‐D‐glucose transport displayed slightly different kinetic parameters. Primary H+ extrusion by the plasma membrane H‐ATPase was not diminished althpough the ATP‐splitting activity was depressed by about 50%. The overall proton‐motive force (pmf) of the fragile mutant at pH 5.5 was only m V while in the parent strain it was 108 m V. In parallel with this, spontaneous acidfication of the external medium to stimulate (a CO2‐associated event) was only about 2% of that in the parent strain. The defect in his, together with the inability to stimulate transport protein synthesis by glucose, may account for the generally poorer transport performance of the fragile mutant.
Cloning and characterization of Baker's yeast α‐glucosidase: Over‐expression in a yeast strain devoid of vacuolar proteinases
Authors: Kopetzki Erhard , Buckel Peter , Schumacher Guenter 
Published: May 2005

AbstractTwo α‐glucosidase (maltase) genes, designated GLUCPI and GLUCPII, have been cloned from an industrial strain of baker's yeast (Saccharomyces cerevisiae) by complementation of a maltase‐negative mutant strain. The different genes were identified according to their alternatively expressed isoenzymes PI and PII in transformants after isoelectric focusing and activity staining in separated cell lysates. The gene encoding α‐glucosidase PI (GLUCPI), which was not present in laboratory strains of S. carlsbergensis with a defined MAL1, 2, 3, 4 or 6 locus, was sequenced and compared with the recently published MAL6S gene. This comparison revealed single amino acid deviations at three positions in the predicted polypeptide sequence. In addition, the divergent promoter region of GLUCPI differed from MAL6S by a triple repeated 147‐bp DNA segment. Maltose induction and glucose repression of α‐glucosidase PI were not affected by the deletion of the repeated DNA segment. However, the absolute expression of α‐glucosidase PI increased two‐ to four‐fold. In addition, a two‐fold increase in the maltase synthesis occurred when the cloned positive regulator gene MAL2‐8cp was on the same plasmid. Furthermore, stability of the α‐glucosidase in cultures in the stationary growth phase was greatly enhanced using a host strain lacking the proteinases A and B and the carboxypeptidases Y and S. Promoter trimming, MAL2‐8cp stimulation and the use of a host strain deficient in four vacuolar proteinases resulted in α‐glucosidase PI expression of about 13% of the soluble protein.
Vanadate inhibition of mitochondrial respiration and H+ ATPase activity in Saccharomyces cerevisiae
Authors: Henderson Gillian E. , Evans Ivor H. , Bruce Ian J. 
Published: May 2005

AbstractThe effects of vandate on mitochondrial respiration and H+ ATPase activity in Saccharomyces cerevisiae were studied. A 50% inhibition of oxygen uptake in isolated mitochondria was produced by 4·4 mM‐V2O5. Activity of H+ ATPase in whole mitochondria was inhibited by 50% by 5·5 μM‐V2O5, in submitochondrial particles by 55 μM‐V2O5; and in the chloroform‐released H+ ATPase by 0·5 mM‐V2O5. Vandate was also found to relieve growth inhibition caused by the mitochondrial H+ ATPase inhibitors NN′‐decyclohexylcarbodiimide and oligomycin. These results imply that vanadate could affect mitochondrial respiration by interacting with the H+ ATPase in S. cerevisiae.
Mutants of the methylotrophic yeast Pichia pinus defective in C2 metabolism
Authors: Tolstorukov I. I. , Efremov B. D. , Benevolensky S. V. , Titorenko V. I. , Sibirny A. A. 
Published: May 2005

AbstractA collection of mutants of Pichia pinus which are unable to grow on ethanol but retain the ability to grow on glucose and methanol, was obtained. Genetic and biochemical analysis of these strains revealed mutations in seven nuclear genes affecting activities of isocitrate lyase (icl1), malate synthase (mls1), phosphoenolpyruvate carboxykinase (pck1), ‘malic’ enzyme (mdd1) and acetyl‐CoA synthetase (acs1, acs2 and acs3). All mutations except acs1‐acs3 have no effect on the activities of other enzymes involved in C2 metabolism. Mutations acs1, acs2 and acs3 have a pleiotropic action, leading to partial reduction in activities of isocitrate lyase and malate synthase. Ethanol‐induced repression of the synthesis of the methanol oxidative enzymes, alcohol oxidase and catalase, is not impaired in these seven mutant classes. On the other hand, C2 compound‐induced inactivation of alcohol oxidase and catalase is impaired in mutants acs1, acs2, acs3 and icl1. It was suggested that glyoxylate and acetate (or acetate precursors) act as low molecular weight effectors, ‘switching on’ inactivation and repression, respectively, of alcohol oxidase and catalase in the medium containing ethanol or acetate.
Functional exploration of the yeast (Saccharomyces cerevisiae) genome: Use of a mini‐mu transposon to analyse randomly cloned DNA sequences
Authors: Daignan‐Fornier Bertrand , Bolotin‐Fukuhara Monique 
Published: May 2005

AbstractThe development of mega‐sequencing techniques requires new methods for global functional analysis of cloned DNA fragments. We have developed a mini‐Mu transposon adapted to yeast cloned DNA fragment analysis. This transposon allows us to do the following in a single construction: (i) to probe yeast cells for the presence of expressed open reading frames (ORFs) in the cloned DNA fragment; (ii) to localize these ORFs in the fragment and determine their transcription orientation; (iii) to use β‐galactosidase protein fusions to study regulation of these ORFs; and (iv) to disrupt the corresponding chromosomal genes.On a 5‐kb yeast DNA sequence, we have verified the reliability of this new tool by comparing the data obtained with the mini‐Mu transposon to those obtained by classical methods.This transposon should be of immediate use in the yeast genome sequencing programme.
Similar and different domains of chitin synthases 1 and 2 of S. cerevisiae: Two isozymes with distinct functions
Authors: Silverman Sanford J. 
Published: May 2005

AbstractSaccharomyces cerevisiae produces two chitin synthases (Chs1 and Chs2) encoded by separate genes. Although these enzymes catalyze the same reaction, Chs2 is essential for septum formation whereas Chs1 has a repair function. To determine if these physiological differences are reflected in the enzyme structures, the CHS2 gene was sequenced and compared to that of CHS1. The predicted amino acid sequence of Chs2 shares substantial similarity with that of Chs1 in the carboxyl two‐thirds of the protein. The amino one‐third segments differ in predicted isoelectric point by almost 5 pH units. It is suggested that the similar regions are related to common catalytic function. The unrelated regions may be involved in regulation or localization of the respective enzymes. CHS1 and CHS2 are unlinked but may have arisen from the duplication of an ancestral gene.
Isolation and characterization of a hexokinase mutant of Schwanniomyces castellii: Consequences on cell production in continuous culture
Authors: Boze Hélène , Moulin G. , Galzy P. 
Published: May 2005

AbstractA mutant of Schwanniomyces castellii with reduced glucose phosphorylation and with practically no phosphorylation of fructose was investigated. Carbon catabolite represion of α‐glucosidase and amylases was reduced. Repression of β‐galactosidase was normal. We have compared in continuous culture this mutant strain with wild type and another previously described mutant. The relationship between the specific rate of glucose consumption (Qs) and residual glucose concentration (s) in an inverse mode, suggests that there may be two types of transport of glucose. Mutation at the phosphorylation level causes apparent modification of the kinetic parameters of glucose uptake rate. The consequence of mutation at the phosphorylation level on biomass production was discussed.
Isolation, DNA sequence and regulation of a new cell division cycle gene from the yeast Saccharomyces cerevisiae
Authors: Hasegawa Hitoshi , Sakai Akira , Sugino Akio 
Published: May 2005

AbstractA new complementation group of temperature‐sensitive mutants of the yeast Saccharomyces cerevisiae (ts26‐1 and ts26‐2) has been isolated and characterized. This mutation maps at 40·7 cM from arg8 and 48·9 cM from arg1 on the left arm of chromosome XV of yeast, providing that it is a newly identified gene. The dumbbell‐shape terminal morphology of the mutant cells at the restrictive temperatures is a characteristic of mutants defective in DNA replication. To study the defect of macromolecule synthesis in the mutant cells, DNA, RNA, and protein synthesis were measured at both permissive and restrictive temperatures. The data suggest that the primary defect of this mutation is at the initiation step of DNA synthesis.The gene has been cloned from an S. cerevisiae genomic library by rescue of the conditional lethality of the mutants. It is present as a single copy in the haploid genome. DNA–RNA hybridization of the gene has identified 1 kb RNA, which is under cell‐division‐cycle control. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of molecular weight 25 055 (214 amino acids).
Isolation and sequence analysis of a K. lactis chromosomal DNA element able to autonomously replicate in S. cerevisiae and K. lactis
Authors: Fabiani Lucia , Aragona Maria , Frontali Laura 
Published: May 2005

AbstractWe have undertaken a search for autonomously replicating (ARSs) from Kluvermyces lactis chromosomal DNA able to sustain plasmid replication in K. lactis and in Saccharomyces cerevisiae. The discovery of such sequences might be interesting for the comparison of ARSs from different sources and possibly useful for the construction of multivalent vectors.HindIII fragments from K. lactis chromosomal DNA were inserted in the YIp5 plasmid (lacking an origin of replication) and the resulting chimaeric plasmids were selected for the ability to transform S. cerevisiae. Four plasmids were identified and further analysed. Two contained the same 1.8 kb K. lactis fragment and transformed both K. lactis and S. cerevisiae with the same efficiency and stability, whereas the third transformed only S. cerevisiae and the fourth transformed K. lactis with a higher efficiency than S. cerevisiae. A detailed study was performed on the 1.8kb fragment which exhibited ARS function in both yeasts. The fragment was subcloned using different restriction enzymes and Bal31 exonuclease. Subclones were tested for ARS function. ARS activities in the two yeasts were localized in the same 100 bp region. Sequencing demonstrated the presence in this region of the dodecanucleotide 5′ATTTATTGTTTT 3′ differing from the ARS core consensus of S. cerevisiae only by a T insertion.A similar nucleotide sequence of S. cerevisiae only in the putative replication origin of the 2 μ‐like plasmid pkD1 which stably replicates in K. lactis. Homologies with ARSs from S. cerevisiae were also found in the regions flanking the above‐mentioned dodecanucleotide.
Liposome‐mediated introduction of proteins into protoplasts of the yeast Hansenula polymorpha as a possible tool to study peroxisome biogenesis
Authors: Douma A. C. , Veenhuis M. , Driessen A. J. M. , Harder W. 
Published: May 2005

AbstractLow pH‐induced fusion between liposomes and protoplasts of the yeast Hansenula polymorpha was demonstrated using a fluorescent assay and freeze‐etch techniques. By this method foreign proteins could be introduced into the cytosol of the protoplast. For instance, after fusion of glucose oxidase‐containing liposomes with protoplasts, activity of this enzyme was demonstrated cytochemically in the cytosol of the resulting protoplasts. Similar results were obtained when ferritin‐containing liposomes were used. Incorporation of foreign proteins into liposomes was not a prerequisite for their introduction into the cytosol of protoplasts. In experiments where ferritin was added to protoplast suspensions together with empty liposomes, this protein was also delivered to the protoplast cytosol. However, in the absence of liposomes no uptake of proteins occurred.We tested the potential of this system in our studies on peroxisome biogenesis. Protoplasts of glucose‐grown H. polymorpha remained stable for prolonged periods in osmotically stabilized cultivation media. Peroxisomes in such protoplasts were capable of protein import and assembly of matrix proteins as was demonstrated by the 20‐fold increase in catalase activity after incubation with methanol or ethanol. However, mature alcohol oxidase purified from H. polymorpha introduced into protoplasts of glucose‐grown cells of this organism was not targeted to peroxisomes as demonstrated with (immuno)cytochemical techniques. The enzyme remained present in the cytosol while its activity gradually decreased. Therefore mature alcohol oxidase probably does not expose the right topogenic signal(s) for recognition by its target organelle.
Creation of ARS activity in yeast through iteration of non‐functional sequences
Authors: Zweifel Stephan G. , L. Fangman Walton 
Published: May 2005

AbstractReplication origins in Saccharomyces cerevisiae have been identified through the clonning of autonomous replication sequence (ARS) elements that allow the extrachromosonal maintenance of plasmid molecules. ARS activity requires a close matcht to an 11 bp consensus sequence and A+T‐rich flanking DNA. ARS elements with a wide range of capacities for promoting plasmid maintenance have been described. We determined the ARS activity of plasmid with inserts consisting of repetitions of a 64 bp 100% A+T sequence that has sequence similarities to known ARS elements. An insert with approximately four repeats did not yield transformants, but inserts with either eight or eleven repeats did. The cooperative of ARS activity did not require a contiguous arrangement since a plasmid containing two inserts of four repeats each, separated by about 1 kb, was functional. Our results show that a charge from non‐function to function can be accomplished by the cumulative action of individually inactive sequences. We conclude that the probability of replication initiation is too low with only four repeats to allow plasmid maintenance, but the overall probability is increased by further sequence iteration to provide origin activity. We suggest that chromosomes may contain streches with dispersed, weak origin elements, each undetected by the conventional ARS assay, that in sum provide origin function.
Analysis of the THR4 region on chromosome III of the yeast Saccharomyces cerevisiae
Authors: Mannhaupt Gertrud , Van Der Linden Gerard , Vetter Irene , Maurer Kick , Pilz Ursula , Planta Rudi , Feldmann Horst 
Published: May 2005

AbstractThe gene encoding theonine synthase (THR4) from the yeast Saccharomyces cerevisiae was cloned by complementation of a thr4 mutant. This gene was also found on a lambda clone (5239) consisting of a fragment of chromosome III inserted in the vector lambda MG3. The THR4 gene encodes a protein of 514 amino acids (M.W. 58 kDa), which has extensive homologies with E. coli threonine synthase (thrC) and B. subtilis threonine synthase. The 5′ flanking region of the gene contains three regulatory sequences. [TGACT(C)] for the general amino acid control (GCN).About 130 bp downstream of the THR4 gene another open reading frame (563 amino acids) is found in the opposite orientation. This may imply that this open reading frame, called CTR86, shares a terminator region with THR4. The function of the protein encoded by CTR86 is not yet clear, but the fact that the upstream region contains a GCN4 responsive site that the gene product may also be involved in amino acid biosynthesis.
The yeast regulatory gene PHO4 encodes a helix‐loop‐helix motif
Authors: Berben Gilbert , Legrain Michèle , Gilliquet Véronique , Hilger François 
Published: May 2005

AbstractEvidence is presented, based on sequence comparisons and secondary structure prediction, of the presence of a DNA‐binding and dimerization helix‐loop‐helix motif in the yeast transcription activator PHO4. Interest in the existence of this first known motif in yeast is discussed.
Ornithine decarboxylase in Saccharomyces cerevisiae: Chromosomal assignment and genetic mapping of the SPE1 gene
Authors: Xie Qiao‐Wen , Tabor Celia White , Tabor Herbert 
Published: May 2005

AbstractThe gene for ornithine decarboxylase in Saccharomyces cerevisiae, SPE1, has been assigned to chromosome XI by the technique of transverse alternating pulsed field electrophoresis and DNA–DNA hybridization. Genetic mapping by tetrad analysis shows that the SPE1 gene is located on the left arm of chromosome XI, 6 cM from the LAP1 gene and 43 cM from the TRP3 gene. The spe10 mutation previously isolated in this laboratory is mapped to the N‐terminal region of the SPE1 gene, and therefore should be designated as a spe1 allele.
Assembly of alcohol oxidase in the cytosol of a peroxisome‐deficient mutant of Hansenula polymorpha—properties of the protein and architecture of the crystals
Authors: Van Der Klei Ida J. , Sulter Grietje J. , Harder Wim , Veenhuis Marten 
Published: May 2005

AbstractWe have studied the expression of alcohol oxidase (AO) in a peroxisome‐deficient mutant strain of Hansenula polymorpha. High levels of octameric, active AO (up to 3·0 U/mg protein) were detected in cells grown at low dilution rates in a glucose‐limited chemostat in the presence of choline as the sole nitrogen source. Monomeric or other intermediate forms of AO were not detected in the mutant strain. This indicated that assembly of the protein into active octameric molecules in the cytosol was as efficient as in wild‐type cells where this process is confined to the peroxisomal matrix. At relatively low rates of expression (less than 1 U/mg protein) AO was localized throughout the cytosol and, surprisingly, was also present inside the nucleus. However, at enhanced levels large crystalloids were formed. Generally one crystalloid was observed per cell, whereas smaller ones were occasionally found in developing buds. Also large crystalloids have been observed inside the nucleus. These crystalloids were not surrounded by a membrane. Based on the morphology of the molecules that constituted these crystalloids and the results of (immuno)cytochemical experiments we conclude that the crystalloids are composed of octameric AO molecules, arranged in a regular lattice, identical to the 3‐dimensional architecture previously described for the crystalline matrix of peroxisomes in methanol‐grown wild type cells of H. polymorpha. Attempts to purify the crystalloids by conventional fractionation methods failed, due to their apparent fragility; however, (immuno)cytochemical experiments revealed that catalase and dihydroxyacetone synthase were also associated with these structures.
Yeast mutants with increased bacterial transposon Tn5 excision
Authors: Gordenin D. A. , Proscyavichus Y. Y. , Malkova A. L. , Trofimova M. V. , Peterzen A. 
Published: May 2005

AbstractFive complementing recessive mutations that exhibit increased bacterial transposon Tn5 precise excision in yeast Saccharomyces cerevisiae were obtained by ethylmethanesulfonate treatment. One of these mutations (tex1) was submitted to extensive genetic analysis. tex1 is a recessive temperature‐sensitive mutation resulting in a 20–100‐fold increase in Tn5 excision. It also has increased frequencies of ochre mutation reversion, of forward mutation to canavanine resistance, and loss of chromosome III or its right arm. The possible mechanism of tex1 effects is discussed.
Influence of the codon following the initiation codon on the expression of the lacZ gene in Saccharomyces cerevisiae
Authors: Looman A. C. , Laude M. , Stahl U. 
Published: May 2005

AbstractA set of 32 different codons were introduced in a lacZ experssion vector (pPTK400) immediately 3′ from the AUG initiation codon. Expression of the lacZ gene was determined in Saccharomyces cerevisiae by measuring the amount of β‐galactosidase fusion protein using immuno‐gel electrophoresis. A 5·3‐fold difference in expression was found among the various constructs. It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression. No correlation could be found between the local secondary structure and expression. When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference. This indicates that in yeast, in contrast to Escherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation. Important determinatnts of the optimal context for an initiation codon in yeast therfore must be located mainly 5′ from this codon.
Applications of high efficiency lithium acetate transformation of intact yeast cells using single‐stranded nucleic acids as carrier
Authors: Gietz R. Daniel , Schiestl Robert H. 
Published: May 2005

AbstractThe highly efficient yeast lithium acetate transformation protocol of Schiestl and Gietz (1989) was tested for its applicability to some of the most important need of current yeast molecular biology. The method allows efficient cloning of genes by direct transformation of gene libraries into yeast. When a random gene pool ligation reaction was transformed into yeast, the LEU2, HIS3, URA3, TRP1 and ARG4 genes were found among the primary transformations at a frequency of approximately 0·1%. The RAD4 gene, which is toxic to Escherichia coli, was also identified among the primary transformants of a ligation library at a frequency of 0·18%. Non‐selective transformation using this transformation proctocol was shown to increase the frequency of gene disruption three‐fold. Co‐transformation showed that 30–40% of the transformation‐competent cells take up more than one DNA molecule which can be used to enrich for integration and delection events 30‐ to 60‐fold. Co‐transformation was used in the construction of simultaneous double gene disruptions as well as disrupting both copies of one gene in a diploid which occurred at 2–5% the frequency of the single event.