bluuebunny/crossref_metadata_2025_split huggingface dataset:
A family of vectors that facilitate transposon and insertional mutagenesis of cloned genes in yeast
Authors: Allen James B. , Elledge Stephen J. 
Published: May 2005

AbstractThis report describes two sets of plasmid vectors that facilitate the identification of regions of complementation in cloned genomic inserts via transposon or insertional mutagenesis. The first set contains ARS‐H4 CEN6, a yeast selectable nutritional marker (HIS3, TRP1 or URA3), and neo for selection in Escherichia coli. These plasmids lack the Tn3 transposition immunity region present in pBR322 derived vectors, and are permissive recipients for Tn3 transposon mutagenesis. The second family of plasmids described facilitate gene disruption procedures performed in vitro. These vectors carry disruption cassettes that contain different yeast selectable markers (HIS3, LEU2, TRP1 or URA3) adjacent to the Tn5 neo gene. These genes can be excised as a cassette on a common restriction fragment and introduced into any desired restriction site with selection for kanamycin resistance.
Genetic and molecular analysis of hybrids in the genus Saccharomyces involving S. cerevisiae, S. uvarum and a new species, S. douglasii
Authors: Hawthorne Don , Philippsen Peter 
Published: May 2005

AbstractWe have studied the phenomenon of infertility of yeast hybrids obtained with physiological conditions under the control of compatible mating systems. The yeasts investigated are three Saccharomyces species: S. cerevisiae, S. uvarum and a new species, S. douglasii. The diploid hybrids from crosses between these species sporulate well but are essentially infertile. The rare viable spores, one per 104 to 105 asci, that have been examined carry a complete genome comprised of chromosomes contributed by both parents but invariably have extra chromosomes, i.e. they are generally disomic for at least two or three chromosomes. This observation is consistent with a failure, in meiosis I, of the pairing and disjunction of homologous chromosomes which in most cases results in spores with an incomplete set of chromosomes. This apparent lack of pairing of ‘homeologous’ chromosomes in meiosis I was analysed in most detail with S. cerevisiae/S. douglasii hybrids. As a genetic tool we studied frequencies of recombination, taking advantage of an S. douglasii breeding stock of some 50 identified mutations in non‐switching haploids. Recombination, although markedly reduced, could be observed at both the chromosomal and allelic levels, implying a sporadic pairing in meiosis to allow genetic exchange. Meiotic recombination frequencies were studied for 14 gene pairs and generally found to be reduced ten‐fold. Heteroallelic recombination (gene conversion) frequencies were measured at 22 loci and were judged to be reduced at least two‐ to 100‐fold. DNA hybridization experiments with S. cerevisiae gene probes gave results consistent with low DNA sequence homologies between S. cerevisiae and S. douglasii. Moreover, by chance, our experiments disclosed another Saccharomyces strain (CBS2908, originally classified as S. cerevisiae) with hybridization patterns identical to S. douglasii except for the hybridization with the Ty transposon probes. Crosses between CBS2908 and S. douglasii yielded diploid hybrids with 80–90% spore viability, thus establishing a second member of the S. douglasii species.
II. Yeast sequencing reports. Analysis of a 70kb region on the right arm of yeast chromosome II
Authors: Mannhaupt Gertrud , Stucka Rolf , Ehnle Susanne , Vetter Irene , Feldmann Horst 
Published: May 2005

AbstractIn the framework of the EC programme for sequencing yeast chromosome II, we have determined the nucleotide sequence of a 70 kb region. Subsequent analysis revealed 35 open reading frames, 14 of which correspond to known yeast genes. From structural parameters and/or similarity searches with entries in the current data libraries, a preliminary functional assessment of several of the putative novel gene products can be made. The gene density in this region amounts to one gene in 1.98 kb. Coding regions occupy 75% of the total DNA sequence. Within the intergenic regions, potential regulatory elements can be predicted. The data obtained here may serve as a basis for a more detailed biochemical analysis of the novel genes. The complete nucleotide sequence of the 70 kb segment as depicted in Figure 1 has been deposited in the EMBL data library under Accession Number X78993.
Efficient expression and secretion of recombinant alpha amylase in Pichia pastoris using two different signal sequences
Authors: Paifer E. , Margolles E. , Cremata J. , Montesino R. , Herrera L. , Delgado J. M. 
Published: May 2005

AbstractWe have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol‐controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2·5 and 0·9 g/l respectively.
Characterization of lipid particles of the yeast, Saccharomyces cerevisiae
Authors: Leber Regina , Zinser Erwin , Paltauf Fritz , Daum Gûnther , Zellnig Gûnther 
Published: May 2005

AbstractLipid particles of the yeast, Saccharomyces cerevisiae, were isolated to high purity and their components were analysed. The hydrophobic core of this organelle consists of triacylglycerols and steryl esters, which are almost exclusively located to that compartment. Lipid particles are stabilized by a surface membrane consisting of phospholipids and proteins. Electron microscopy confirmed the purity of the preparations and the proposed structure deduced from biochemical experiments. Major proteins of lipid particles have molecular weights of 72, 52, 43 and 34 kDa, respectively. The 43 kDa protein reacts with an antiserum against human apolipoprotein AII. In lipid particles of the yeast mutant strain S. cerevisiae erg6, which is deficient in sterol Δ24‐methyltransferase, this protein is missing thereby identifying the protein and confirming our previous finding (Zinser et al., 1993) that sterol Δ24‐methylation is associated with lipid particles. A possible involvement of surface proteins of lipid particles in the interaction with other organelles is discussed with respect to sterol translocation in yeast.
Isolation and preliminary characterization of Pichia pinus mutants insensitive to glucose repression
Authors: Alamäe Tiina , Simisker Jaan 
Published: May 2005

AbstractA new method for the isolation of glucose repression‐insensitive mutants in the methylotrophic yeast Pichia pinus was developed. The method is based on screening of small suspension samples derived from 2‐deoxyglucose‐resistant colonies for alcohol oxidase activity. Alcohol oxidase activity was evaluated by determination of formaldehyde excreted by cells. Mutants with glucose non‐repressible alcohol oxidase and catalase synthesis were obtained. All mutants grew poorly on D‐xylose compared to the wild type, whereas growth on L‐arabinose was similar to the wild type. Changes in the glucose transport system were suggested to be responsible for altered growth characteristics and defective glucose repression.
Near‐Stoichiometric interaction between the non‐specific lipid‐transfer protein of the yeast Candida tropicalis and peroxisomal acyl‐coenzyme A oxidase prevents the thermal denaturation of the enzyme in vitro
Authors: Niki Toshiro , Bun‐Ya Masanori , Muro Yoshitaka , Kamiryo Tatsuyuki , Hiraga Yoshikazu 
Published: May 2005

AbstractA 14‐kDa peroxisomal‐matrix protein, named PXP‐18, of the yeast Candida tropicalis is a structural and functional homologue of the mammalian nonspecific lipid‐transfer protein (identical to sterol carrier protein‐2). PXP‐18 protected acyl‐coenzyme A oxidase (ACO), the rate limiting enzyme of the peroxisomal β‐oxidation of fatty acids, from thermal inactivation at 48°C or 70°C. This effect was dose‐dependent and not replaceable either by chicken egg white lysozyme, which is similar to PXP‐18 (insofar as it is basic, small, and monomeric), or by bovine serum albumin, a carrier of lipids in the blood. ACO was irreversibly denatured by heat treatment at 70°C for 15 min. However, when ACO and PXP‐18 were similarly heat‐treated, they formed a large complex at a molar ratio of PXP‐18 to ACO subunit that was about one, independent of their initial ratio. This near‐stoichiometric complex had ACO activity after a 500‐fold dilution and was accompanied by ACO that was free of PXP‐18 and indistinguishable from native ACO in size and activity. PXP‐18 also protected urate oxidase, another peroxisomal enzyme, from inactivation at 66°C for 15 min and facilitated the renaturation of ACO denatured by 2 M urea. These results indicated that PXP‐18 is active in modulating the structure of peroxisomal enzymes in vitro. It is possible that PXP‐18 functions as a stress protein or as a part of the system that keeps peroxisomal proteins intact.
IX. Yeast sequencing reports. Cloning and sequence of REV7, a gene whose function is required for DNA damage‐induced mutagenesis in Saccharomyces cerevisiae
Authors: Torpey Laurence E. , Gibbs Peter E. M. , Nelson John , Lawrence Christopher W. 
Published: May 2005

AbstractThe function of the REV7 gene is required for DNA damage‐induced mutagenesis in budding yeast, Saccharomyces cerevisiae, and is therefore thought to promote replication past sites of mutagen damage in the DNA template. We have cloned this gene by complementation of the rev7‐2 mutant defect, and determined its sequence. REV7 encodes a predicted protein of Mr 28 759 which is unlike any other protein in the NCBI non‐redundant protein sequence data base, and which is inessential for viability. The sequence of the 3·88 kb yeast genomic fragment containing REV7 has been deposited in Genbank accession number U07228.
II. Yeast sequencing reports. A 12·5 kb fragment of the yeast chromosome II contains two adjacent genes encoding ribosomal proteins and six putative new genes, one of which encodes a putative transcriptional factor
Authors: Démolis Nadine , Mallet Laurent , Jacquet Michel 
Published: May 2005

AbstractThe nucleotide sequence of a 12·5 kb fragment localized to the right arm of chromosome II of Saccharomyces cerevisiae has been determined. The sequence contains eight putative genes. Two of them are contiguous and represent two ribosomal protein genes: SUP46 and URP1. SUP46 is implicated in translation fidelity and encodes the ribosomal protein S13. URP1 is homologous to the rat ribosomal protein gene L21. The open reading frame (ORF) YBR1245 is similar in its N‐terminal part to transcription factors like SRF and MCM1. The ORF YBR1308 shows homology with proteins of the AAA‐family (ATPases Associated with diverse cellular Activities). Two genes are predicted to encode putative membrane proteins. The sequence has been deposited in the EMBL data library under Accession Number U02073.
Consideration of the evolution of the Saccharomyces cerevisiae MEL gene family on the basis of the nucleotide sequences of the genes and their flanking regions
Authors: Turakainen Hilkka , Kristo Paula , Korhola Matti 
Published: May 2005

AbstractAnalysis of the DNA sequences of new members of the Saccharomyces cerevisiae MEL1‐MEL10 gene family showed high homology between the members. The MEL gene family, α‐galactosidase‐coding sequences, have diverged into two groups; one consisting of MEL1 and MEL2 and the other of MEL3‐MEL10. In two S. cerevisiae strains containing five or seven MEL genes each, all the genes are nearly identical, suggesting very rapid distribution of the gene to separate chromosomes. The sequence homology and the abrupt change to sequence heterogeneity at the centromere‐proximal 3′ end of the MEL genes suggest that the distribution of the genes to new chromosomal locations has occurred partly by reciprocal recombination at solo delta sequences.We identified a new open reading frame sufficient to code for a 554 amino acid long protein of unknown function. The new open reading frame (Accession number Z37509) is located in the 3′ non‐coding region of MEL3‐MEL10 genes in opposite orientation to the MEL genes (Accession numbers Z37508, Z37510, Z37511). Northern analysis of total RNA showed no hybridization to a homologous probe, suggesting that the gene is not expressed efficiently if at all.
The linkage of (1–3)‐β‐glucan to chitin during cell wall assembly in Saccharomyces cerevisiae
Authors: Hartland Robbert P. , Vermeulen Cees A. , Sietsma Johannes H. , Wessels Joseph G. H. , Klis Frans M. 
Published: May 2005

AbstractPulse‐chase experiments with [14C]glucose demonstrated that in the cell wall of wild‐type Saccharomyces cerevisiae alkali‐soluble (1–3)‐β‐glucan serves as a precursor for alkali‐insoluble (1–3)‐β‐glucan. The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali‐insoluble glucan. These cells, unable to form a glucan–chitin complex, were highly vulnerable to hypo‐osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall.Conversion of alkali‐soluble glucan into alkali‐insoluble glucan occurred both early and late during budding and also in the ts‐mutant cdc24‐1 in the absence of bud formation.
Molecular analysis of the SNF8 gene of Saccharomyces cerevisiae
Authors: Yeghiayan Paula , Tu Jiangling , Vallier Laura G. , Carlson Marian 
Published: May 2005

AbstractMutations in the SNF8 gene impair derepresson of the SUC2 gene, encoding invertase, in response to glucose limitation of Saccharomyces cerevisiae. We report here the cloning of the SNF8 gene by complementation. Sequence analysis predicts a 26 936‐dalton product. Disruption of the chromosomal locus caused a five‐fold decrease in invertase derepression, defective growth on raffinose, and a sporulation defect in homozygous diploids. Genetic analysis of the interactions of the snf8 null mutation with spt6/ssn20 and ssn6 suppressors distinguished SNF8 from the groups, SNF1, SNF4 and SNF2, SNF5, SNF6. Notably, the snf8 ssn6 double mutants were extremely sick. Mutations of SNF8 and SNF7 showed similar phenotypes and genetic interactions, and the double mutant combination caused no additional phenotypic impairment. These findings suggest that SNF7 and SNF8 are functionally related. The complete nucleotide sequence of SNF8 has been deposited in GenBank under accession number U10361.
Sequence, mapping and disruption of CCC2, a gene that cross‐complements the Ca2+‐sensitive phenotype of csg1 mutants and encodes a P‐type ATPase belonging to the Cu2+‐ATPase subfamily
Authors: Fu Dadin , Beeler Troy J. , Dunn Teresa M. 
Published: May 2005

AbstractWe have isolated, sequenced, mapped and disrupted a gene, CCC2, from Saccharomyces cerevisiae. This gene displays non‐allelic complementation of the Ca2+‐sensitive phenotype conferred by the csg1 mutation. Analysis of the CCC2p amino acid sequence reveals that it encodes a member of the P‐type ATPase family and is most similar to a subfamily thought to consist of Cu2+ transporters, including the human genes that mutate to cause Wilson disease and Menkes disease. The ability of this gene, in two or more copies, to reverse the csg1 defect suggests that Ca2+‐induced death of csg1 mutant cells is related to Cu2+ metabolism. Cells without CCC2 require increased Cu2+ concentrations for growth. Therefore CCC2p may function to provide Cu2+ to a cellular compartment rather than in removal of excess of Cu2+. The sequence of CCC2 is available through GenBank under accession number L36317.
Transient responses of Candida utilis to oxygen limitation: Regulation of the Kluyver effect for maltose
Authors: Kaliterna Janko , Weusthuis Ruud A. , Castrillo Juan I. , Van Dijken Johannes P. , Pronk Jack T. 
Published: May 2005

AbstractThe facultatively fermentative yeast Candida utilis exhibits the Kluyver effect for maltose: this disaccharide is respired and assimilated but, in contrast to glucose, it cannot be fermented. To study the mechanism of the Kluyver effect, metabolic responses of C. utilis to a transition from aerobic, sugar‐limited growth to oxygen‐limited conditions were studied in chemostat cultures. Unexpectedly, the initial response of maltose‐grown cultures to oxygen limitation was very similar to that of glucose‐grown cultures. In both cases, alcoholic fermentation occurred after a lag phase of 1 h, during which glycerol, pyruvate and D‐lactate were the main fermentation products. After ca. 10 h the behaviour of the maltose‐ and glucose‐grown cultures diverged: ethanol disappeared from the maltose‐grown cultures, whereas fermentation continued in steady‐state, oxygen‐limited cultures grown on glucose. The disappearance of alcoholic fermentation in oxygen‐limited chemostat cultures growing on maltose was not due to a repression of the synthesis of pyruvate decarboxylase and alcohol dehydrogenase. The results demonstrate that the Kluyver effect for maltose in C. utilis does not reflect an intrinsic inability of this yeast to ferment maltose, but is caused by a regulatory phenomenon that affects a key enzyme in maltose metabolism, probably the maltose carrier. The observed kinetics indicate that this regulation occurs at the level of enzyme synthesis rather than via modification of existing enzyme activity.
Nucleotide sequence and characterization of the Saccharomyces cerevisiae RPL19A gene encoding a homolog of the mammalian ribosomal protein l19
Authors: Song Jae Mahn , Cheung Edwin , Rabinowitz Jesse C. 
Published: May 2005

AbstractA gene designated RPL19A has been identified in the region downstream from the 3′‐end of the Saccharomyces cerevisiae MIS1 gene encoding the mitochondrial C1‐tetrahydrofolate synthase. The gene codes for the yeast ribosomal protein YL19 which exhibits 57·5% identity with the mammalian ribosomal protein L19. RPL19A is one of two functional copies of the YL19 gene located on chromosome II. The disruption of RPL19A has no effect on the growth of the yeast. The RPL19A gene contains an intron located near the 5′‐end. The 5′‐flanking region contains one similar and one complete UASrpg upstream activating sequence. RPL19A was also found to be adjacent to the chromosome II AAC3 gene, encoding the mitochondrial ADP/ATP carrier protein. The nucleotide sequence(s) reported in this paper has been submitted to the GenBanktm/EMBL data bank with the accession number Z36751.
Regulation of carbon metabolism in chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol
Authors: De Jong‐Gubbels Patricia , Vanrolleghem Peter , Heijnen Sef , Van Dijken Johannes P. , Pronk Jack T. 
Published: May 2005

AbstractGrowth efficiency and regulation of key enzyme activities were studied in carbon‐ and energy‐limited chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol at a fixed dilution rate. Biomass yields on substrate carbon and oxygen could be adequately described as the net result of growth on the single substrates. Activities of isocitrate lyase and malate synthase were not detected in cell‐free extracts of glucose‐limited cultures. However, both enzymes were present when the ethanol fraction in the reservoir medium exceeded the theoretical minimum above which the glyoxylate cycle is required for anabolic reactions. Fructose‐1,6‐bisphosphatase activity was only detectable at high ethanol fractions in the feed, when activity of this enzyme was required for synthesis of hexose phosphates. Phospho‐enol‐pyruvate‐carboxykinase activity was not detectable in extracts from glucose‐grown cultures and increased with the ethanol fraction in the feed. It is concluded that, during carbon‐limited growth of S. cerevisiae on mixtures of glucose and ethanol, biosynthetic intermediates with three or more carbon atoms are preferentially synthesized from glucose. Synthesis of the key enzymes of gluconeogenesis and the glyoxylate cycle is adapted to the cells′ requirement for these intermediates. The gluconeogenic enzymes and their physiological antagonists (pyruvate kinase, pyruvate carboxylase and phosphofructokinase) were expressed simultaneously at high ethanol fractions in the feed. If futile cycling is prevented under these conditions, this is not primarily achieved by tight control of enzyme synthesis.
Sequence analysis of a 33·1 kb fragment from the left arm of Saccharomyces cerevisiae chromosome X, including putative proteins with leucine zippers, a fungal Zn(II)2‐Cys6 binuclear cluster domain and a putative α2‐SCB‐α2 binding site
Authors: Miosga T. , Schaaff‐Gerstenschläger I. , Chalwatzis N. , Baur A. , Boles E. , Fournier C. , Schmitt S. , Velten C. , Wilhelm N. , Zimmermann F. K. 
Published: May 2005

AbstractIn the framework of the European BIOTECH project for sequencing the Saccharomyces cerevisiae genome, we have determined the nucleotide sequence of the left part of the cosmid clone 232 and the cosmid clone 233 provided by F. Galibert (Rennes Cedex, France). We present here 33,099 base pairs of sequence derived from the left arm of chromosome X of strain S288C. This sequence reveals 17 open reading frames (ORFs) with more than 299 base pairs, including the published sequences for ARG3, LIGTR/LIG1, ORF2, ACT3 and SCP160. Two other ORFs showed similarity with S. cerevisiae genes: one with the CAN1 gene coding for an arginine permease, and one with genes encoding the family of transcriptional activators containing a fungal Zn(II)2‐Cys6 binuclear cluster domain like that found in Ppr1p or Gal4p. Both putative proteins contain a leucine zipper motif, the Can1p homologue has 12 putative membrane‐spanning domains and a putative α2‐SCB‐α2 binding site. In a diploid disruption mutant of ORF J0922 coding for the transcriptional activator homologue, no colonies appeared before 10 days after transformation and then grew slowly. In contrast, haploid disruption mutants showed a growth phenotype like wild‐type cells. One ORF showed weak similarity to the rad4 gene product of Schizosaccharomyces pombe and is essential for yeast growth. Five ORFs showed similarity to putative genes on the right arm of chromosome XI of S. cerevisiae. Two of them have similarity to each other and belong to a family of extracellular proteins that groups mammalian SCP/Tpx‐1, insects Ag3/Ag5, plants PR‐1 and fungi Sc7/Sc14. Three small ORFs are completely contained in the larger ORFs on the corresponding complementary strand and thus probably do not represent real genes. The DNA sequence has been deposited in the EMBL data library under Accession Number X83502.