bluuebunny/crossref_metadata_2025_split huggingface dataset:
Extraction and rapid inactivation of proteins from Saccharomyces cerevisiae by trichloroacetic acid precipitation
Authors: Wright Anthony P. H. , Bruns Michael , Hartley Brian S. 
Published: May 2005

AbstractMethods currently used for the extraction of proteins from yeast involve relatively long time periods between sampling cells from a culture and analysis of their proteins by polyacrylamide gel electrophoresis–sodium dodecylsulphate. Often it is desirable to inactivate cellular metabolism rapidly after sampling and here we show that trichloroacetic acid precipitation techniques, often used for rapid extraction and inactivation of proteins from higher eukaryotes, can be adapted for use with organisms which have cell walls.
Sequence and mutational analysis of ESS1, a gene essential for growth in Saccharomyces cerevisiae
Authors: Hanes Steven D. , Shank Peter R. , Bostian Keith A. 
Published: May 2005

AbstractA newly isolated gene, ESS1, was shown to encode a protein required for vegetative growth in Saccharomyces cerevisiae. The nucleotide sequence of ESS1 revealed a 172 amino acid open reading frame predicting a highly basic, 19·5 kilodalton product. Although the gene was isolated by cross‐hybridization with the vertebrate v‐sis oncogene, the primary amino acid sequence bears only a slight resemblance to the p28sis protein. ESS1 was shown to be single copy in the yeast genome and transcriptionally active during logarithmic growth. It is located on the right arm of chromosome X, 6 centimorgans distal to ilv3. The genetic map location indicates it is not allelic to any previously characterized mutation in this organism. Both inactivation of ESS1 by gene disruption and overexpression by fusion to a heterologous promoter were detrimental to growth in both haploid and diploid cell types. Under non‐permissive conditions, the terminal phenotype of strains containing a suppressible amber mutation within ESS1 was one of aberrant multibudded structures. Examination of this morphology indicates that loss of ESS1 function may lead to a defect in cytokinesis or cell separation.
Size distribution and general structural features of N‐linked oligosaccharides from the methylotrophic yeast, Pichia pastoris
Authors: Grinna Lynn S. , Tschopp Juerg F. 
Published: May 2005

AbstractThe secreted glycoproteins of Pichia pastoris contain more than 35% of their N‐linked oligosaccharides as structures smaller than Man14GlcNAc2 (Man = mannose; GlcNAc = N‐acetylglucosamine). On heterologous invertase produced in P. pastoris, approximately 85% of the oligosaccharides are in the size range Man8–14GlcNAc2. The structures appear to contain α‐linked mannose. In addition, one‐third of the structures contain net negative charge and can be radio‐labelled in vivo with 32P. The largest oligosaccharides isolated from P. pastoris are significantly shorter than the hypermannosylated structures typical of S. cerevisiae, indicating that the factors which influence the processing of N‐linked oligosaccharides in P. pastoris are different from those which influence processing in S. cerevisiae. The smaller N‐linked oligosaccharides synthesized by P. pastoris resemble high‐mannose oligosaccharides synthesized by animal cells, and this finding increases the utility of P. pastoris as a host for the production of heterologous glycoproteins.
Two pathways of DNA double‐strand break repair in G1 cells of Saccharomyces cerevisiae
Authors: Glasunov Alexander V. , Glaser Vadim M. , Kapultsevich Yuri G. 
Published: May 2005

AbstractG1 cells of the diploid yeast Saccharomyces cerevisiae are known to be capable of a slow repair of DNA double‐strand breaks (DSB) during holding the cells in a non‐nutrient medium (Luchnik et al., 1977; Frankenberg‐Schwager et al., 1980). In the present paper, S. cerevisiae cells γ‐irradiated in the G1 phase of the cell cycle are shown to be capable of fast repair of DNA DSB; this process is completed within 30‒40 min, of holding the cells in water at 28°C. For this reason, the kinetics of DNA DSB repair during holding the cells in a non‐nutrient medium are biphasic, i.e., the first ‘fast’ phase is completed within 30‒40 min, whereas the second, ‘slow’ phase is completed within 48 h. Mututions rad51, rad52, rad54 and rad55 inhibit the fast repair of DNA DSB, whereas mutations rad50, rad53 and rad57 do not significantly influence this process.It has been shown that the observed fast and slow repair of DNA DSB in the G1 diploid cells of S. cerevisiae are separate pathways of DNA DSB repair in yeast.
5′‐Secondary structure formation, in constrast to a short string of non‐preferred codons, inhibits the translation of the pyruvate kinase mRNA in yeast
Authors: Bettany Andrew J. E. , Moore Paul A. , Cafferkey Robert , Bell Les D. , Goodey Andrew R. , Carter Bruce L. A. , Brown Alistair J. P. 
Published: May 2005

AbstractThe effects of poor codon bias and secondary structure formation upon the translation of the pyruvate kinase (PYK1) mRNA have been investigated in Saccharomyces cerevisiae. Following insertion mutagenesis at the 5′‐end of the PYK1 coding region, the gene was transformed into yeast, and translation assessed directly in vivo by determining the distribution of the modified PYK1 mRNAs across polysomes fractionated by sucrose density gradient centrifugation. The chromosomally‐encoded (wild‐type) PYK1 mRNA, and the actin, ribosomal protein L3 and glyceraldehyde‐3‐phosphate dehydrogenase mRNAs were used to control for minor differences between polysome preparations. An insertion containing 13 non‐preferred codons at the 5′‐end of the coding region was found to have no significant effect upon PYK1 mRNA translation. In contrast, translation was inhibited by an insertion which increased the formation of secondary structures at the 5′‐end of the mRNA (overall ΔG = −36·6 kcal/mol). Control insertions were also analysed to exclude the possibility that alterations to the amino acid sequence of pyruvate kinase affect the translation of its mRNA. These insertions, which introduced preferred codons or restored wild‐type levels of secondary structure formation, did not significantly influence PYK1 mRNA translation.
Cloning and sequencing of the gene for apocytochrome b of the yeast Kluyveromyces lactis strains WM27 (NRRL Y‐17066) and WM37 (NRRL Y‐1140)
Authors: Brunner Aurora , Coria Roberto 
Published: May 2005

AbstractThe apocytochrome b genes from two strains of the yeast Kluyveromyces lactis, have been isolated and sequenced. The coding sequences in strains WM27 (NRRL Y‐17066) and WM37 (NRRL Y‐1140) were identical but the upstream noncoding regions were slightly different. The sequences demonstrated the presence of a continuous open reading frame with no introns. The amino acid sequence, derived from the coding strand, showed 82% homology to the apocytochrome b of Saccharomyces cerevisiae strain D273–10B and only 58% homology to the protein from Schizosaccharomyces pombe strain 50. CUN and CGN codon families were absent from the K. lactis gene. Codon usage was very similar to that of other mitochondrial genomes with mostly U or A in the third position. There were two unusual features. All threonines were coded by ACA(U) and all arginines by AGA.
Characterization of the adr1‐1 nonsense mutation identifies the translational start of the yeast transcriptional activator ADR1
Authors: Bemis Lynne T. , Denis Clyde L. 
Published: May 2005

AbstractWe have characterized a nonsense mutation in the ADR1 gene that identifies the translational start of the ADR1 protein. The ADR1 gene of Saccharomyces cerevisiae is required for synthesis of the glucose‐repressible alcohol dehydrogenase (ADH2). The adr1‐1 mutation, which inhibits ADH2 expression, was identified as a C to G transversion at base pair +32. This alteration would result in a UGA nonsense codon in place of a serine codon that would lead to termination of the ADR1 polypeptide after the 10th amino acid. The effect of the adr1‐1 mutation was partially reversed by UGA‐tRNA suppressors, indicating that the adr1‐1 mutation affects ADR1 expression at the translational level. These observations establish that the first available AUG in the ADR1 sequence is used as the translational start site of ADR1. Tyrosine or leucine UGA‐tRNA‐suppressors resulted in levels of adr1‐1 activity similar to that found for a serine UGA‐tRNA‐suppressor, suggesting that serine residue‐11 is not essential to ADR1 function. Northern analyses showed that the 5·1 kb ADR1 mRNA was two‐ to three‐fold more abundant when isolated from a strain carrying the ADR1 allele than from an isogenic strain containing the adr1‐1 allele. These data confirm that the 5·1 kb mRNA is the ADR1 mRNA and suggest that inhibition of adr1‐1 mRNA translation results in more rapid degradation of the adr1‐1 mRNA.
Physiology of mutants with reduced expression of plasma membrane H+‐ATPase
Authors: Vallejo Carmen G. , Serrano Ramon 
Published: May 2005

AbstractTwo mutations containing insertions and deletions in the promoter in the plasma membrane H+‐ATPase gene (PMA1) of Saccharomyces cerevisiae have been introduced into the genome by homologous recombination, replacing the wild‐type gene. The resulting strains have 15 and 23% of the wild‐type ATPase content. Decreased levels of ATPase correlate with decreased rates of proton efflux and decreased uptake rates of amino acids, methylamine, hygromycin B and tetraphenylphosphonium. This supports a central role of the enzyme in yeast bioenergetics. However, the final accumulation gradient of tetraphenylphosphonium is not affected by the mutations and that of methylamine and 2‐aminoisobutyric acid is only decreased in the most extreme mutant. Apparently, kinetic constraints seem to prevent the equilibration of yeast active transports with the electrochemical proton gradient. As expected from their transport defects, the ATPase‐deficient mutants are more resistant to hygromycin B and more sensitive to acidification than wild‐type yeast. Mutant cells are very elongated, suggesting a structural role of the ATPase in the yeast surface.
Occurrence of peroxisomal membrane proteins in methylotrophic yeasts grown under different conditions
Authors: Sulter G. J. , Looyenga L. , Veenhuis M. , Harder W. 
Published: May 2005

AbstractWe have the substructure and polypeptide composition of the peroxisomal membranes in two methylotrophic yeasts in relation to different growth conditions. The results obtained that no significant ultrastructural differences existed between the membranes of variously grown cells.The presence of specific peroxisomal membrane proteins (PMPs) was studied biochemically. On sodium dodecyl sulphate‐polyacrylamide gels of purified microbody membranes isolated from methanol‐grown Hansenula polymorpha, prominent proteins bands were observed at 22, 31, 35, 42, 49 and 51 kD. These proteins were also present when the cells were grown in media containing ethanol and/or ethylamine. Apart from these, several other PMPs were specifically induced under these conditions, namely 24, 29, 37 and 62 kD proteins. The polypeptide composition of peroxisomal membranes from H. polymorpha was compared with that of another methylotroph. Candida biodinii. In the latter organism a specific PMP with a molecular weight of 23 kD was induced during growth on D‐alanine instead of ammonium sulphate as the nitrogen source.
Immunocytochemical demonstration of the peroxisomal ATPase of yeasts
Authors: Douma A. C. , Veenhuis M. , Waterham H. R. , Harder W. 
Published: May 2005

AbstractThe presence of an ATpase on yeast peroxisomal membranes was studied by immunological methods. Western blot analysis of purified peroxisomal membranes from several yeasts revealed distinct cross‐reaction with specific antibodies against the F1‐part or the β‐subunit of the mitochondrial ATpase of Saccharomyces cerevisiae. This was not due to mitochondrial contamination as was demonstrated by analytical sucrose gradient centrifugation.Protein A‐gold labelling carried out on Lowicryl‐embedded methanol‐grown Hansenula polymorpha using these antibodies did not result in significant staining. However, when organelles isolated from this yeast were successively incubated with antibodies and protein A‐gold prior to embedding, specific labelling was observed on both the peroxisomal membrane and the membrane of damaged mitochondria but not on intact mitochondira. Specific labelling of the peroxisomal membrane was confirmed by freeze‐fracture immunocytochemistry. In addition to the peroxisomal membrane, the mitochondrial membrane was also labelled in these experiments. Freeze‐fracture immunocytochemistry was also successful for the localization of peroxisomal matrix proteins, e.g. alcohol oxidase and dihydroxyacetone synthase and of mitochondrial membrane proteins, e.g. cytochrome c oxidase.
Comparative study on cytochrome P‐450 of yeasts using specific antibodies to cytochromes P‐450alk and P‐45014DM
Authors: Kaergel E. , Aoyama Y. , Schunck W.‐H. , Mueller H.‐G. , Yoshida Y. 
Published: May 2005

AbstractThe occurrence of cytochrome P‐45014DM(lanosterol 14α‐demethylase) and cytochrome P‐450alk (long‐chain alkane terminal hydroxylase) in various yeast strain was determined with immunological procedures. Cytochrome P‐45014DM, which is a constitutive or housekeeping enzyme playing an essential role in ergosterol biogenesis, was found in all yeast strains so far tested. Cytochromes P‐45014DM from different species of yeast were immunologically different, although they may have a few common antigenic sites. In contrast, cytochrome P‐450alk was detected only in the alkane‐assimilating yeasts.
A K2 neutral Saccharomyces cerevisiae strain contains a variant K2 M genome
Authors: Wingfield Brenda D. , Southgate Valerie J. , Pretorius Isak S. , Van Vuuren Hendrik J. J. 
Published: May 2005

AbstractK2 neutral strain Saccharomyces cerevisiae USM12 was identified and characterized. This strain carried an M double‐stranded RNA (dsRNA) genome encoding for resistance to K2 toxin. The M dsRNA was larger than the K2 killer yeast M dsRNA and homoduplex analysis of denatured and reannealed K2 neutral M dsRNA revealed an inverted duplication. Heteroduplex analysis showed that two thirds of the K2 M genome had homology with K2 neutral M genome. Hybridization showed that the USM12 M dsRNA has significant homology with the K2M dsRNA. Protein profiles of extracellular proteins from USMA12 and cured strain indicated that USM12 did not secrete any toxin. This is the first time that a K2 neutral yeast strain has been characterized.
Gaba transport in Saccharomyces cerevisiae
Authors: McKelvey Joyce , Rai Rajendra , Cooper Terrance G. 
Published: May 2005

AbstractGamma‐aminobutyrate (GABA) accumulation in growing cultures of Saccharomyces cerevisiae was shown to occur by means of an active transport system that is inhibited by proton ionophores, azide, fluoride and arsenate ions. Transport occurred maximally at pH 5·0 and exhibited apparent Km values of 12 μM and 0·1 mM. Accumulated GABA did not efflux upon treatment with proton ionophores and exchanged with extracellular material only very slowly. However, release was complete upon treatment with nystatin. These observations raise the possibility that a major portion of intracellular GABA is sequestered in the vacuole. The response of GABA uptake to growth on various nitrogen sources suggested that uptake may be subject to several types of regulation.
An essential gene in Saccharomyces cerevisiae shares an upstream regulatory element with PRP4
Authors: Petersen‐Bjørn Sara , Harrington T. R. , Friesen James D. 
Published: May 2005

AbstractORF2 is an essential gene immediately upstream of PRP4 (formeryl RNA4), a gene involved in nuclear mRNA processing in Saccharomyces cerevisiae. The two genes are arranged head‐to‐head. An 8 base‐pair conserved sequences element is found upstream of both genes, as well as upstream of certain other genes that are known to be involved in pre‐mRNA processing. Through deletion analysis we have found that both of the conserved sequence elements are important for transcription of both genes. We have cloned ORF2 and have isolated temperature‐sensitive orf2 mutants. The phenotype of these mutants does not suggest a role for ORF2 in mRNA processing. The deduced amino acid sequence of ORF2 indicates significant similarity to DPR1, a gene encoding a protein that is involved in the carboxy‐terminal processing of G‐protein.
Characterization of the cytochrome c gene from the starch‐fermenting yeast Schwanniomyces occidentalis and its expression in bakers's yeast
Authors: Amegadzie Bernardy Y. , Zitomer Richard S. , Hollenberg Cornelis P. 
Published: May 2005

AbstractA cytochrome c protein gene, CYC10, of the dextran‐ and starch‐fermenting yeast, Schwanniomyces occidentalis was cloned and characterized. The DNA sequence was determined, and the predicted amino acid sequence of the protein‐coding region shares close homologies to the cytochrome c genes. A. S. occidentalis strain with a disruption of the gene revealed that CYC10 was the only functional cytochrome c protein‐encoding gene in S. occidentalis, unlike the two cytochrome c protein genes (CYC1 and CYC7) in Saccharomyces cerevisiaear The CYC10 gene was oxygen‐induced but not subject to catabolite repression.The expression of the CYC10 gene was studied in the heterologous yeast. S. cerevisiae. The oxygen induction of the gene was found to be identical to that of the CYC1 gene, indecating these two genes share similar or closely related cis‐ and trans‐ acting oxygen regulatory elements. However, the CYC10 gene was glucose repressed in S. cerevisiae strains; a phenomenon which was not observed in the native S. accidentalis cells. Search in the 5′ unstranslated region of the CYC10 gene revealed some homologies at −425 to −405 to UAS1 of the S. cerevisiae CYC1 gene. A deletion of a segment of upstream region including this sequence abolished expression in S. cerevisiae.Finally the phylogenetic relationships of different yeasts and fungi were determined based upon the amino acid sequences of the cytochrome c proteins. These relationships do not completely agree with classical divisions.
The omnipotent suppressor SUP45 affects nucleic acid metabolism and mitochondrial structure
Authors: Pocklington Michael J. , Johnston Lee , Jenkins John R. , Orr Elisha 
Published: May 2005

AbstractYeast (Saccharomyces cerevisiae) strains sensitive to a variety drugs were used to select for novobiocin‐resistant mutants that were simultaneously temperature‐sensitive. The mutants remained as sensitive as the parent strains to a wide range of drugs other than novobiocin, and did not exhibit any suppression of suppressible auxotrophic markers. At the non‐permissive temperature, the mutant cells arrested mainly as unbudded cells, and were instantly defective in DNA and RNA synthesis, but not protein synthesis. The cloned wild‐type gene was identified as SUP45, which has been previously implicated in the translation process. Our results suggest that SUP45 may have a function in addition to, or different from, the one that has been assigned to it previously.
Effect of leader primary structure on the translational efficiency of phosphoglycerate kinase mRNA in yeast
Authors: Van Den Heuvel Joop J. , Planta Rudi J. , Raué Hendrik A. 
Published: May 2005

AbstractIn order to determine the effect of nucleotide composition of the 5′‐untranslated (leader) region on the translational efficiency of mRNA in yeast, we replaced a large part of the leader region of the phosphoglycerate kinase (PGK) gene by various deoxyoligonucleotides of defined sequence. All mutations left the context of the transcription initiations site and AUG start codon intact. The mutant genes were introduced into yeast cells on a multicopy vector and the ratio of the steady‐state levels of PGKmRNA and protein were determined.We found the translational efficiency to be unaffected by the presence of either an 18 nucleotides (nt) long poly A or poly C tract or by sequences consisting of mixtures of A and C residues in any proportion. In contrast, a polyU tract, as well as mixtures of U and C residues, reduced translational efficiency by a factor of two to three, presumably by long‐rang base ‐pairing between the leader and sequences elsewhere in the coding or 3′‐non‐coding regions of the messenger. In agreement with this hypothesis, a five‐fold reduction in translational efficiency was found for an mRNA carrying a polyC tract in the leader as well as a polyG tract in the trailer, neither of which had any effect on translational efficiency by itself. Therefore, we conclude that the leader and trailer regions (including the polyA tail) of PGK mRNA are sufficiently close to base‐pair when containing complementary sequences. The resulting secondary structure evidently constitutes a barrier for incoming 40S subunits on their way to the AUG start codon.The presence of an 18 nt long polyG tract in the leader completely abolished translation of the PGK mRNA in accordance with earlier observations. However, we found the leaders containing up to 40% G residues interspersed with either A or U, still allow highly efficient translation. This value is about four times as high as the average G content of leader sequence in naturally occurring yeast mRNAS.Finally, neither deletion of about 40% of the trailer sequence of PGK mRNA, not replacement of this sequence by homopolymer tracts had any effect on translational efficiency.
Evaluation of phylogenetic relationships among fission yeast by nDNA/nDNA reassociation and conventional taxonomic criteria
Authors: Martini Ann Vaughan 
Published: May 2005

AbstractThe genus Schizosaccharomyces comprises a small, somewhat heterogeneous group of yeast species which have in common a unique mode of vegetative reproduction by cross‐wall formation without constriction as well as a certain degree of osmophilia, most strains having been isolated from habitats of high sugar concentration. This study evaluated inter‐ and intraspecific relationships utilizing nDNA/nDNA optical reassociation and by the analysis of physiological profiles of several strains of each species. Results demonstrate that the genus should be divided into three species: Schiz. japonicus, Schiz. octosporus and Schiz. pombe.
Increased endocytosis in the Saccharomyces cerevisiae fragile mutant VY1160
Authors: Waltschewa L. , Kotyk A. , Venkov P. 
Published: May 2005

AbstractThe VY1160 mutant is characterized by cell lysis in hypotonic solutions and generally increased permeability to substances for which Saccharomyces cerevisiae cells are not permeable. Two mutations, srb1 and ts1, have been identified in VY1160 mutant, and previous studies (Kozhina et al., 1979) have shown srb1 to be responsible for cell lysis. We now present evidence that the ts1 mutation leads to increased endocytosis in VY1160 cells. The internalization of lucifer yellow carbohydrazide in VY1160 cells is time‐, temperature‐ and energy‐dependent an consistent with a fluid‐phase mechanism of endocytosis. The rate of steady‐state accumulation of hte dye at 37°C is 145 ng/μg DNA per h for VY1160 mutant and 23 ng/μg DNA per h for S288C parental strain. Studies with isogenic strains having either the srb1 or the ts1 mutation, or SRB1 TS1 wild‐type alleles have shown that only ts1 strains possess increased endocytosis. Quantitation of endocytosis in cells grown at 24°C and shifted at 38°C shows that ts1 strains, but not srb1 and wild‐type strains, increase ten‐fold the internalization of lucifer yellow 2 h after the shift at 38°C. The analysis of ts1 × wild‐type crosses provides evidence that the temperature‐sensitive phenotype segregates together with the enhanced endocytosis. It is concluded that the increased endocytosis might the generally increased permeability of VY1160 mutant cells.