bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha
Authors: Janowicz Zbigniew A. , Melber Karl , Merckelbach Armin , Jacobs Eric , Harford Nigel , Comberbach Martin , Hollenberg Cornelis P.
Published: May 2005

An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co‐express both the L (preS1‐S2‐S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol‐inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5–8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle‐specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H. polymorpha‐derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.
Expression of the α‐galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha
Authors: Fellinger Arthur J. , Verbakel John M. A. , Veale Rosemary A. , Sudbery Peter E. , Bom Isaac J. , Overbeeke Nico , Verrips C. Theo
Published: May 2005

The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the α‐galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 μm plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the α‐galactosidase is controlled by the methanol‐regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the α‐galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active α‐galactosidase enzyme was efficiently secreted (>85%) and after methanol induction, the expression level was 42 mg/l. Amino‐terminal sequencing of the purified α‐galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted α‐galactosidase was glycosylated and had a sugar content of 9·5%. The specific activity of the α‐galactosidase produced by H. polymorpha was 38 U mg−1 compared to 100 U mg−1 for guar α‐galactosidase. Deglycosylation of the H. polymorpha α‐galactosidase restored the specific activity completely.
The role of pyruvate decarboxylase in the Kluyver effect in the food yeast, Candida utilis
Authors: Sims A. P. , Stålbrand H. , Barnett J. A.
Published: May 2005

The glucose‐fermenting yeast, Candida utilis cannot use the β‐D‐glucoside, cellobiose, anaerobically, although it is able to do so aerobically. β‐Glucoside transport and hydrolysis and pyruvate decarboxylase activities of this yeast were measured aerobically and anaerobically. β‐Glucoside transport was five‐fold faster aerobically than anaerobically, but there was no corresponding difference in β‐glucosidase activity. Pyruvate decarboxylase activity varied greatly, being synthesized de novo in response to the presence of D‐glucose and anaerobic conditions and about 50% deactivated on the removal of D‐glucose or the addition of air. Activation and deactivation were rapidly reversible. Failure to utilize cellobiose anaerobically, in particular, and the Kluyver effect, in general, probably depends on much reduced glycolytic flux, associated under anaerobic conditions, with (i) lower transport rate, (ii) low substrate affinity of the relevant glycosidase and (iii) deactivation of pyruvate decarboxylase. So, in addition to the complex effects of oxygen, anaerobiosis and specific sugars on induction, repression and derepression, there are fine controls on pyruvate decarboxylase activity, leading to fast activation or deactivation of the enzyme.
TDH2 is linked to MET3 on chromosome X of Saccharomyces cerevisiae
Authors: Mountain Harry A. , Korch Christopher
Published: May 2005

The MET3 gene of Saccharomyces cerevisiae was cloned and its restriction map was found to differ in the upstream region from an earlier published map (Cherest et al. Gene 34, 269–281, 1985) and nucleotide sequence (Cherest et al. Mol. Gen. Genet. 210, 307–313, 1987). Southern blot analysis of genomic DNA from strains S288C and FL100 (the genetic backgrounds from which these different copies of the gene had been cloned) showed that our clone from a S288C‐based library had the same restriction map as the chromosomal DNA from both of the strains. Comarison of the nucleotide sequence of the two clones indicated that the earlier published clone probably represented a cloning artifact. In our clone, we found upstream of MET3, the nucleotide sequence of the TDH2 gene (Holland and Holland, J. Biol. Chem. 255, 2596–2605, 1980). The chromosomal orientation of the two genes was determined to be MET3–TDH2–CEN10.
The sequence of a 6·3 kb segment of yeast chromosome III reveals an open reading frame coding for a putative mismatch binding protein
Authors: Valle Giorgio , Bergantino Elisabetta , Lanfranchi Gerolamo , Carignani Giovanna
Published: May 2005

We report the sequence of a 6·3 kb segment of DNA mapping near the end of the right arm of chromosome III of Saccharomyces cerevisiae. The sequence reveals a major open reading frame coding for a putative protein of 1047 amino acids with a striking similarity to the bacterial proteins involved in recognition of mismatched DNA base pairs. This is particularly interesting as the existence of a yeast mismatch repair system similar to that of bacteria has been postulated for some years, but a yeast protein homologous to the bacterial mismatch binding protein had not been identified.

The results of a comparison of the putative yeast mismatch binding protein with the bacterial mismatch binding proteins and with two cognate mammalian sequences, support the idea that a similar mismatch repair system may be present also in mammalian cells. The possibility that all of these proteins may have evolved from a common ancestral gene is also discussed.
Yeast flocculation: Reconciliation of physiological and genetic viewpoints
Authors: Stratford Malcolm
Published: May 2005

Yeast floccultion results from surface expression of specific proteins (lectins). Two flocculation phenotypes were suggested by physiological and biochemical tests, whereas genetic data suggested a larger number of mechanisms of flocculation. After reviewing the biochemistry, physiology and genetics of flocculation, a new hypothesis combining the data available from these different sources, is proposed.

Flocculation results when lectins present on flocculent cell bind sugar residues of neighbouring cell walls. These sugar receptors are intrinsic to the mannan comprizing cell walls of Saccharomyces cerevisiae. Two lectin phenotypes were revealed by sugar inhibition studies. The gluco‐ and mannospecific NewFlo phenotype is not, as yet, found in genetically defined strains. Mannospecific flocculation (Flo 1 phenotype) is found in strains containing the genes FLO1, FLO5 and FLO8. This phenotype is also found following mutation of the TUP1 or CYC8 loci, in previously non‐flocculent strains. It is therefore proposed that the structure gene for mannospecific flocculation is common or possibly unbiquitous in non‐flocculent strains and in consequence, FLO1, FLO5 and FLO8 are probably regulatory genes, exerting positive control over the structure gene.

Flocculation expression requires lectin secretion to the cell surface. Many of the observed ‘suppressions’ of flocculation may be due to mutations of the secretory process, involved in transporting structural proteins to the cell wall.

The possible involvement of killer L double‐stranded RNA with flocculation is suggested, given the lectin properties of viral coat proteins nad an association between L double‐stranded RNA and the Flo 1 phenotype.
The small heat‐shock protein Hsp26 of Saccharomyces cerevisiae assembles into a high molecular weight aggregate
Authors: Bentley Nicola J. , Fitch Ian T. , Tuite Mick F.
Published: May 2005

Hsp26 is one the major small heat‐shock proteins (Hsp) of the Saccharomyces cerevisiae. yet its cellular role remains to be discovered. To examine the cellular consequences of overexpression of Hsp26, the gene encoding this protein (HSP26) was overexpressed from a multicopy plasmid using either its own promoter or by coupling it to the effecient constitutive PGK promoter. The PGK promoter provided the opportunity to overexpress Hsp26 under nonstress conditions and such high level synthesis, prior to a lethal heat shock (50°C), gave a small but reproducible elevation in thermotolerance. In transformed strains overexpressing Hsp26 under either stressed or non‐stress conditions, the Hsp26 polypeptide was recovered almost exclusively as a high molecular weight aggregate. This high molecular weight aggregate (or heat‐shock granule, HSG) was purified by differential centrifugation and sucrose gradient density centrifugation and shown, by electron microscopic analysis, to be of a uniform size (15–25 nm diameter). Analysis of the purified HSG demonstrated that it had a molecular weight of 550 kDa, yet contained no other integral polypeptides or other macromolecules.
Sequence of a 10·7 kb segment of yeast chromosome XI identifies the APN1 and the BAF1 loci and reveals one tRNA gene and several new open reading frames including homologs to RAD2 and kinases
Authors: Jacquier Alain , Legrain Pierre , Dujon Bernard
Published: May 2005

We report here the DNA sequence of a segment of chromosome XI of Saccharomyces cerevisiae extending over 10·7 kb. The sequence was determined using a double‐strand sequencing strategy adapted from the random‐clone strategy. The segment contaiins seven non‐overlapping long open reading frames, YKL500, 505, 510, 513, 516, 518 and 520 and one tRNA gene. YKL505 and YKL513 are two already sequenced genes, the BAF1/ABF1 and the APN1 genes, respectively. YKL510 exhibits a strong homology to the RAD2 protein and YKL516 is presumabloy a protein kinase.
Identification of a novel secreted glycoprotein of the yeast Saccharomyces cerevisiae stimulated by heat shock
Authors: Lupashin V. V. , Kononova S. V. , Ratner Ye. N. , Tsiomenko A. B. , Kulaev I. S.
Published: May 2005

A new secreted yeast glycoprotein with an Mr of about 400 kDa (gp400) has been found. The glycoprotein is an O‐mannosylated oligomer, whose synthesis and export into culture medium are stimulated by heat shock. Intracellular transport of gp400 is carried out by membrane vesicles distinct form the known constitutive scretory vesicles. Immunological analysis revealed gp400 only in Saccharomyces species.
Molecular cloning and physical analysis of an 8·2 kb segment of chromosome XI of Saccharomyces cerevisiae reveals five tightly linked genes
Authors: Abraham Philip R. , Mulder Anita , Riet Jan Van'T , Planta Rudi J. , Raué Hendrik A.
Published: May 2005

The nucleotide sequence of 6472 base pairs of an 8·2 kb segment of Saccharomyces cerevisiae chromosome XI has been determined. The sequence contains a cluster of four long open reading frame (ORF) designated YKL2, YKL3, YKL4 and TGL1 in the same orientation, flanked at the 5′‐end by a divergent incomplete ORF (YKL1). Transcription and Southern analyssis of the four complete ORFs showed that all are expressed and are present in single copy on the haploid genome. The average codon adaption index of the coding regions is approximately 0·2, suggesting that these genes are lowly expressed. The upstream regions of all four genes as well as the YKL1 ORF contain putative promoter elements previously found to be characteristic of nuclear genes encoding mitochondrial proteins. Significant sequence similarities were found between the YKL3 protein and Escherichia coli ribosomal protein S2 as well as between the TGL1 protein and triglyceride lipases from rat salivary gland and human gastric tissue. The 3′‐end of the 6472 bp nucleotide sequence overlaps with the upstream region of the previously identified CTK1 gene, encoding the largest subunit of CTD kinase (Lee, J. M. and Greenleaf, A. L., 1991, Gene Expression 2, 149–167), thereby increasing the number of genes on the 8·2 kb fragment to at least five. The transcripts of these genes represent approximately 83% of the DNA fragment, making it one of the most highly transcribed regions of the yeast chromosome analysed to date.
Molecular cloning and analysis of autonomous replicating sequence of Candida maltosa
Authors: Sasnauskas Kestutis , Jomantienè Rasa , Lebedienè Edita , Lebedys Jurgis , Januška Albinas , Janulaitis Arvydas
Published: May 2005

A Candida maltosa chromosomal DNA fragment which confers high frequency transformation of C. maltosa and autonomous replication of recombinant plasmids was cloned and sequenced. Analysis of the nucleotide sequence of the cloned DNA revealed a sequence homologous for C. maltosa autonomously replicating sequence (ARS) elements. Vector pRJ1 for C. maltosa was constructed, which contained a 1.3 kb ARS sequence, pICEM‐19H and the ADE1 gene of C. maltosa. Southern blot analysis suggested that the copy number of pRJ1 in C. maltosa was approximately 20 per genome. The sequence analysis also revealed an open reading frame, encoding a polypeptide with high homology (70%) to the RS15 protein of Brugia pagangi. This open reading frame has an intron with canonical sites for correct splicing in Saccharomyces cerevisiae.
N‐linked glycosylation of proteinase B precursors of the yeast Saccharomyces cerevisiae is not require for proper targeting or processing of the enzyme
Authors: Nebes Vicki L. , Jones Elizabeth W.
Published: May 2005

Proteinase B precursors are modified by an N‐linked carbohydrate side chain at Asn 314. Glycosylation at this position is not required for proper localization, processing, or activation of the enzyme.
SEC6 encodes an 85 kDa soluble protein required for exocytosis in yeast
Authors: Potenza Marc , Bowser Robert , Müller Heike , Novick Peter
Published: May 2005

The SEC6 gene encodes a protein required for an event leading to fusion of post‐Golgi vesicles with the plasma membrane in Saccharomyces cerevisiae cells. The gene was cloned by complementation of the temperature‐sensitive growth defect of a sec6‐4 strain. The nucleotide sequence was determined and the longest open reading frame was found to encode an 85 kDa protein of 733 amino acids. The Sec6 protein is predicted to be hydrophilic and is found predominantly in the soluble fraction of a yeast lysate, in a species that sediments with a coefficient of 14S. No extnsive homology was found with known proteins of the database. Gene disruption and marker rescue experiments indicate that SEC6 is a single copy gene essential for growth. Overproduction of Sec6p does not suppress any of the other lateactind sec mutants, yet sec6‐4 does display synthetic lethality with sec8‐9, suggesting that the two products may fulfill inter‐related functions.
Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly
Authors: Gould Stephen J. , McCollum Dannel , Spong Allan P. , Heyman John A. , Subramani Suresh
Published: May 2005

We describe the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas). These mutants of P. pastoris can be identified solely by their inability to grow on methanol and oleic acid, the utilization of which requires peroxisomal enzymes, and are defined by the absence of normal peroxisomes as judged by electron microscopy and biochemical fractionation experiments. These mutants are the result of genetic defects at single loci and represent at least eight different complementation groups. The isolation of pas mutants of P. pastoris by a simple screen for mutants unable to use methanol and oleic acid represents a significantly more efficient method for identification of pas mutants than is possible in other organisms. To exploit this advantage fully we also developed new reagents for the genetic and molecular manipulation of P. pastoris. These include a set of auxotropic strains with an essentialiy wild type genetic background, plasmids that act as Escherichia coli–P. pastoris shuttle vectors, and genomic DNA libraries for isolation of P. pastoris genes by functional complementation of mutants or by nucleic acid hybridization. The availability of numerous pas mutants and the reagents necessary for their molecular analysis should lead to the isolation and characterization of genes involved in peroxisome assembly.
Mutational analysis of Schizosaccharomyces pombe U4 snRNA by plasmid exchange
Authors: Dandekar Thomas , Tollervey David
Published: May 2005

We have developed a system for testing mutations by plasmid exchange in the fission Schizosaccharomyces pombe. This system has been used to test the requirement for different regions of the small nuclear RNA U4 in S. pombe. Surprisingly, five of seven deletion and substitution mutations tested in different regions of U4 prevent the accumulation of the mutant RNA. Substitution of the U4 sequence in stem 1 of the U4/U6 interaction doamain allows accumuation of the mutant U4, but does not supports viability. Two sequences with homology to the Sm binding site are found in the 3′ region of S. pombe U4; substitution of the 3′ sequence of the two does not interfere with accumulation or function of U4, indicating that the 5′ sequence is the functional Sm‐binding site.
The telomere‐associated MAL3 locus of Saccharomyces is a tandem array of repeateed genes
Authors: Michels Corinne A. , Read Evan , Nat Karyl , Charron Maureen J.
Published: May 2005

Saccharomyces strains capable of fermenting maltose contain any one of five telomere‐associated MAL loci. Each MAL locus is a complex of three genes encoding the three functions required to ferment maltos: maltose permease (GENE 1), maltase (GENE 2) and the MAL trans‐activator (GENE 3). All five loci have been cloned and all are highly sequence homologous over least a 9‐0 kbp region containing these GENEs (Charron et al., Genetics 122, 307–331, 1989). Our initial studies of strians carrying the MAL3 locus indicated the presence of linked, repeated MLA‐homologous sequences (Michels and Needleman, Mol. Gen. Genet. 191, 225–230, 1983). Here we report our analysis of the centromere‐proximal MAL3‐linked sequence and show that the complete MAL3 locus spans approximately 40 kbp and consists of tandemly arrayed, partial repeats of the three GENE sequences described above. In addition, the structure of the MAL3 locus is compared to that of three partially functional alleles of MAL3. These alleles were shown to contain only MAL31 and MAL32 and their structure suggests that they resulted from MAL3 deletions removing the sequences centromere‐proximal of MAL31. The amplification and rerrangement of the telomer‐linked MAL3 sequences are discussed in the context of studies on other telemere‐associated sequences from yeast and other species.
MOL1, a Sacchromyces cerevisiae gene that is highly expressed in early stationary phase during growth on molasses
Authors: Praekelt U. M. , Meacock P. A.
Published: May 2005

We have isolated a new Saccharamyces cerevisiae gene, MOL1, that is transiently expressed at high levels in the early stationary phase of batch cultures growing on industrial molasses medium. The DNA sequence of the M O L I gene (for MOLasses‐inducible) with its flanking regions was determined (EMBL accession number X61669). It encodes a polypeptide of M, 35 kDa that is closely related to stress‐inducible proteins of similar size frow two Fusarium species. Unlike ST135 of Fusarium, MOL1 is not induced by ethanol or heat shock. MOL1 expression is absent in rich (YP) medium, and only very low levels of expression are detectable in minimal (YNB) medium. The gene is not essential, and a MOL1 distruption strain showed no apparent phenotype undera variety of growth conditions.

The 5′ region of MOL1 contains the complete sequence previously determined for the SUF4 locus, encoding a tRNA‐gly (UCC) gene, which has been mapped to chromosome VII.
II. Yeast sequencing reports. Sequence of a 12·7 kb segment of yeast chromosome II identifies a PDR‐like gene and several new open reading frames
Authors: Delaveau TH. , Jacq C. , Perea J.
Published: May 2005

A 12,684 bp DNA fragment, between FUS3 and the centromere, from the left arm of chromosome II of Saccharomyces cerevisiae was sequenced as part of the European project to sequence the whole chromosome. This segment contains at least five complete new open reading frames (ORFs) and the beginning (191 first 5′ codons) of an ORF whose putative translational product is highly similar to the multidrug resistance PDR1 gene previously characterized by Balzi et al. (1987) on chromosome VII.
Schizosaccharomyces pombe mitochondria: Morphological, respiratory and protein import characteristics
Authors: Moore Anthony L. , Walters Andrew J. , Thorpe Julian , Fricaud Anne‐Catherine , Watts Felicity Z.
Published: May 2005

A technique is described for the isolation and purification of intact, respiratory‐competent mitochondria from Sachizo‐saccharomyces pombe. The purified mitochondria are capabel of oxidizing NADH and succinate as resporatory substrates. indicating the presence of succinate dehydorgenase and an NADH dehydrogenase located on the outer suface of the inner membrane. Mitochondria display good respiratory control with an ADP/O ratio of <2. Respiratory activity is linearly dependent upon the redox poise of the quinone pool, suggesting the presence of an unbranched repiratory pathway to molecular oxygen. Immunogold labelling using antisera raised against mitochodria proteins (SSPI, SSCI, and PHSPI) from three different species, namely S. pombe, Saccharomyces cerevisiane and the plant Pisum sativum respectively, has been used to investigate the presence and ultrastructure of the mitochondria isolated by this procedure. The immunocytochemistry was carried out using cells containing wild‐type levels of SSPI protein and cells over‐expressing the protein. These results also demonstrate the capacity of mitochondria to import increased levels of protein in vivo. In vitro import experiments using COXIV‐DHFR indicate that purified S. pombe mitochondria can efficiently import this precursor, and that protein translocation is dependent upon an oxidizable substrate and a membrane potential.
XI. Yeast sequencing reports. The sequence of a 9·3 kb segment located on the left arm of the yeast chromosome XI reveals five open reading frames including the CCE1 gene and putative products related to MYO2 and to the ribosomal protein L10
Authors: Pascolo Steve , Ghazvini Marjan , Boyer Jeanne , Colleaux Laurence , Thierry Agnès , Dujon Bernard
Published: May 2005

We report the wequence of a 9·3 kb DNA segment of chromosome XI of Saccharomyces cerevisiae, located between the MAK11 locus and the centromere. This sequence contains four long open reading frames (ORFs). YKL160, YKL162, YKL164, YKL165 and part of another ORF, YKL166, covering altogether 90% of the entire sequence. One of these ORFs YKL164, corresponds to CCE1. Translation products of two other ORFs, YKL160 and YKL165, exhibit homology with previously known S. cerevisiae proetins: the robosomal protein L10, and the MYO2 gene product, respectively.
RNA delivery in Saccharomyces cerevisiae using electroporation
Authors: Everett Jeffrey G. , Gallie Daniel R.
Published: May 2005

An efficient delivery method for introducing in vitro synthesized RNA into yeast into has been developed using electroporation. Spheroplast preparation, electroporation, and subsequent expression analysis can be accomplished within a single day. The use of introduced mRNA constructs avoids any complications due to nuclear regulation and is particularly suited for cytoplasmic regulatory studies. Moreover, this technique is useful for introduicing those RNas that connot be made in vivo, Such as poly (A)−mRNAs or RNAs with base modifications. We demonstrate that the Escherichia coli GUS gene and the firefly Luc gene are both excellent reporter genes for RNA electroporation.
Cloning and disruption of a gene required for growth on acetate but not on ethanol: The acetyl‐coenzyme a synthetase gene of Saccharmoyces cerevisiae
Authors: De Virgilio Claudio , Bürckert Niels , Barth Gerold , Neuhaus Jean‐Marc , Boller Thomas , Wiemken Andres
Published: May 2005

A DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl‐coenzyme A (acetyl‐CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromsome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79·2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons dowunstream, is in a more conventional context. Possible implications of two alternative traslational start sites for the celular lacalization of the enzyme are disussed. A stable mutant of this gene, obtained by the gene disruptiona technique, had the same low basal acitivity of acetyl‐CoA synthetase as wild‐type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetlyl‐CoA synthetase (ACSI) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction os isocitrate lyase on acetate media, indicating that activity of acetyl‐CoA sytheatase is dispensable for incuctionof the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACSI gene did not affect growth on media containing ethanol as the sole crbon source. Demonstrating that threre are alternative pathways pathways leading to acetyl‐CoA under these condintions.
Fluorocytosine causes uncoupled dissipation of the proton gradient and behaves as an imperfect substrate of the yeast cytosine permease
Authors: Hopkins P. , Shaw R. , Acik L. , Oliver Simon , Eddy A. A.
Published: May 2005

At‐pH 5–6 ATP‐depleted washed cell preparations of strain NC233‐10b[pII4‐9], in which the cytosine permease was overexpressed, absorbed cytosine, hypoxanthine or fluorocytosine stoichiometrically with, respectively, about 1, 1·4 and 5 proton equivalents. The cellular pH fell proportionately. The membrane depolarization caused by each compound was assayed in the presence of glucose with a voltage‐sensitive dye and increased in the same order. Fluorocytosine significantly lowered the growth yield that a ‘petite’ strain of the yeast formed at limiting glucose concentrations. At pH 5·6 with extracellular [K+] below 1 mM, each of the three substrates was accumulated about 200‐fold from a dilute solution at the expense of the proton gradient. This concentration ratio corresponds to a solute gradient (Δμs) of 13 kJ mol−1. Raising [K+]0 systematically lowered the substrate accumulation ratio and ΔμH. The mean ratio Δμs/ΔμH was 0·82 all three substrates. It was concluded that whereas the behaviour of cytosine approximated to that expected for a symport of unit proton stoichiometry, the absorption of protons with fluorocytosine and, to a lesser extent, hypoxanthine, was only partly conserved as useful work. A possible mechanism of this novel phenomenon is outlined.
Analysis of glucose repression in Saccharomyces cerevisiae by pulsing glucose to a galactose‐limited continuous culture
Authors: Sierkstra Laurens N. , Nouwen Nico P. , Verbakel John M. A. , Verrips C. Theo
Published: May 2005

In this study, glucose repression in Saccharomyces cerevisiae was analysed under defined physiological conditions, at both the molecular and physiological levels, by pulsing glucose to a galactose‐limited continuous culture. During this pulse of glucose, the galactose feed was kept constant. Directly after the glucose pulse, carbon dioxide production increased while oxygen consumption remained constant, demonstrating that the surplus of glucose had been consumed by means of fermentation. The direct accumulation of galactose in the medium after the glucose pulse indicated that the consumption of galactose had been stopped instantaneously. Galactose uptake experiments revealed that the galactose transporter was still present but apparently was incapable of galactose uptake, which could be due to inhibition of the galactose transporter by glucose. The total concentration of cAMP increased from 5 nmol g−1 at t = 0 to 25 nmolg−1 at t = 1·5 min. After 2 min the concentration of cAMP gradually decreased again to the normal level. Within 2 min after the addition of glucose, the transcription of the GAL genes and SUC2 was inhibited. In addition, the transcription of the HXK1 gene, encoding hexokinase isoenzyme 1, was also inhibited, which demonstrates that the HXK1 gene is regulated at the transcriptional level comparable with invertase.
Functional expression of bacterial β‐glucuronidase and its use as a reporter system in the yeast Yarrowia lipolytica
Authors: Bauer Ronald , Paltauf Fritz , Kohlwein Sepp D.
Published: May 2005

The use of β‐glucuronidase (β‐GUS) as a reporter and sensitive detection system for Yarrowia lipolytica was studied. The Escherichia coli gusA gene was expressed under control of the homologous LEU2 promoter in a transcriptional fusion. An NcoI restriction site was introduced at the translational start‐ATG, conserving the most favorable context for initiation of translation. The chimeric LEU2′‐gusA gene was integrated into the LEU2 locus by homologous recombination. The β‐GUS assay was very sensitive and highly reproducible, using the cytosolic fraction or a total cell extract as the source of enzyme. In a leucine‐free medium, β‐GUS activity was at a high, constant level, independent of growth phase. In transformants grown on complete medium, β‐GUS activity was reduced about three‐fold, but doubled during logarithmic growth. No intrinsic β‐GUS activity was detectable in untransformed Y. lipolytica and no effect of β‐GUS expression on growth was obseved. β‐GUS‐producing Y. lipolytica cells could be directly detected on media plates containing X‐gluc (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐glucuronide).
QCR9, the nuclear gene encoding a small subunit of the mitochondrial cytochrome bc1 complex, maps to the right arm of chromosome VII in Saccharomyces cerevisiae
Authors: Phillips John D. , Trumpower Bernard L.
Published: May 2005

We present here mapping data for QCR9, a nuclear gene encoding a subunit of the ubiquinol‐cytochrome c oxidoreductase complex. Deletion of QCR9 results in the inability of cells to grow on non‐fermentable carbon sources at 37°C. Thus, qcr9 mutants can be scored by growing cells on YPE/G at 37°C, or followed by the URA3 marker, which was inserted when making the qcr9 deletion strain, JDP1. The location of QCR9 on the right arm of chromosome VII with respect to the previously mapped genes ADE3, SER2 and PET54 is given.
The complete sequence of a 19,482 bp segment located on the right arm of Chromosome II from Saccharomyces cerevisiae
Authors: Doignon Francois , Biteau Nicolas , Crouzet Marc , Aigle Michel
Published: May 2005

We report here the sequence of a 19,482 bp DNA segment of chromosome II of Saccharomyces cerevisiae. The fragment contains 16 open reading frames (ORFs) covering 74% of the sequence. Four predicted products present homology with known proteins. The ORF YBR1732 exhibits a strong homology to serine hydroxymethyl transferase; the best score is 53·1% identity in 458 amino acids overlap with the serine hydroxymethyl transferase from rabbit liver. YBR1724, which shows homology with riboflavin synthase of Bacillus subtilis, is probably the RIB5 gene implied in riboflavine synthesis and mapped in this region. YBR1733 is homologous to rab protein and YBR1728 is presumably a GTPase activating protein.
Sequence of a cytochrome c gene from Kluyveromyces lactis and its upstream region
Authors: Picos M. Angeles Freire , Torres Ana M. Rodriguez , Ramil Elvira , Cerdan M. Esperanza , Breunig Karin D. , Hollenberg Cornelis P. , Zitomer Richard S.
Published: May 2005

The complete sequence of a cytochrome c gene from Kluyveromyces lactis including its upstream region is reported. Sequence of the translated open reading frame is discussed in terms of cytochrome c structural requirements. Putative regulatory signals in the upstream region are described and compared with reported sequences which modulate the expression of respiratory‐related yeast genes.
Molecular genetics in Saccharomyces kluyveri: The HIS3 homolog and its use as a selectable marker gene in S. kluyveri and Saccharomyces cerevisiae
Authors: Weinstock Keith G. , Strathern Jeffrey N.
Published: May 2005

We cloned the Saccharomyces kluyveri HIS3 homolog, k‐HIS3, and made a partial deletion of the gene. The k‐HIS3 gene complemented a HIS3 deletion in S. cerevisiae. The DNA sequences of the open reading frames (ORFs) of the HIS3 homologs are 70% identical at the DNA level and 83% identical at the deduced amino acid level. The ORF upstream of the k‐HIS3 gene is related to the PET56 gene of S. cerevisiae found upstream of the HIS3 gene of S. cerevisiae. The ORF downstream from the k‐HIS3 gene is not related to the DED1 gene found downstream of the HIS3 gene in S. cerevisiae.
Physical localization of yeast CYS3, a gene whose product resembles the rat γ‐cystathionase and Escherichia coli cystathionine γ‐synthase enzymes
Authors: Barton Arnold B. , Kaback David B. , Clark Michael W. , Keng Teresa , Ouellette B. F. Francis , Storms Reg K. , Zeng Bin , Zhong Wuwei , Fortin Nathalie , Delaney Shawn , Bussey Howard
Published: May 2005

We have cloned, sequenced and physically mapped the CYS3 gene of Saccharomyces cerevisiae. This gene can complement the cys3–1 allele, and disruptions at this locus lead to cysteine auxotrophy. The predicted CYS3 product is closely related (46% identical) to the rat cystathionine γ‐lyase (Erickson et al., 1990), but differs in lacking cysteine residues. These results provide further evidence that the S288C strain of yeast resembles mammals in synthesizing cysteine solely via a trans‐sulfuration pathway. The CYS3 product was found to have strong homology to three other enzymes involved in cysteine metabolism: the Escherichia coli metB and metC products and the S. cerevisiae MET25 gene product. The trans‐sulfuration enzymes appears to form a diverged family and carry out related functions from bacteria to mammals.
Secretion of mouse α‐amylase from fission yeast Schizosaccharomyces pombe: Presence of chymostatin‐sensitive protease activity in the culture medium
Authors: Tokunaga Masao , Kawamura Akiko , Yonekyu Sayuri , Kishida Masao , Hishinuma Fumio
Published: May 2005

We have constructed two secretion vectors for Schizosaccharomyces pombe using an SV40 promoter and the secretion signals of the pGKL killer toxin complex derived from Kluyveromyces lactis. Although indigenous secretory glycoproteins tend to accumulate in the periplasmic space of S. pombe, we have succeeded in the secretion of mouse α‐amylase into the culture medium. The efficiency of secretion, processing pattern, stability and culture conditions for mouse α‐amylase were studied in S. pombe. The 128 kDa killer secretion signal was more effective in directing secretion of mouse α‐amylase than the 28 kDa killer secretion signal. We detected a chymostatin‐sensitive protease activity in the culture medium of S. pombe, which digests mouse α‐amylase secreted into the culture medium. The addition of 5 μg/ml chymostatin was shown to protect mouse α‐amylases from this degradation.
Rapid identification and characterization of peroxisomal assembly mutants in Yarrowia lipolytica
Authors: Nuttley William M. , Brade Anthony M. , Eitzen Gary A. , Glover John R. , Aitchison John D. , Rachubinski Richard A. , Gaillardin Claude
Published: May 2005

We describe the isolation and characterization of peroxisomal assembly mutants in the genetically manipulable yeast Yarrowia lipolytica (pay mutants). These mutants were initially identified as oleic acid‐non‐utilizers by their inability to grow on oleic acid, the utilization of which requires peroxisomal β‐oxidation enzymes. Identification of a subset of oleic acid‐non‐utilizers as pay mutants was obtained by a rapid immunofluorescence procedure using antibodies to the peroxisomal targeting signal Ser‐Lys‐Leu‐CO2H. Punctate structures characteristic of peroxisomes were not detected in pay mutants using this technique. This rapid identification by immunofluorescence should be generally applicable to the selection of peroxisomal assembly mutants in other yeasts. To take advantage of the pay mutant system, we constructed a genomic library in the autonomously replicating vector pINA445 and developed an efficient and rapid electroporation procedure for the functional complementation of these mutants. We have been successful in functionally complementing two independent pay mutants. Molecular analysis of these and other complementing genes will allow for characterization of some of the cellular elements involved in peroxisomal assembly.
Increased transformation levels in intact cells of Saccharomyces cerevisiae aculeacin A‐resistant mutants
Authors: Gallego Carme , Casas Celia , Herrero Enrique
Published: May 2005

Several Saccharomyces cerevisiae mutants resistant to the cell wall synthesis inhibitor aculeacin A exhibit higher transformation levels than the parental strain. Mutant acr2 has been studied in more detail. It is transformed up to ten‐fold more efficiently than the wild‐type strain with episomal, centromeric and integrative plasmids, and dimethyl sulfoxide has an additive effect improving transformation efficiency. Transformation with linear DNA molecules is not as much affected in acr2 cells. The observed effects may be caused by the altered plasma membrane composition of the mutants.
Absence of cell wall chitin in Saccharomyces cerevisiae leads to resistance to Kluyveromyces lactis killer toxin
Authors: Takita Marco A. , Castilho‐Valavicius Beatriz
Published: May 2005

Kluyveromyces lactis killer toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations in Saccharomyces cerevisiae that leads to toxin resistance and a temperature‐sensitive phenotype. These mutant cells arrest growth at 37°C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to be CAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23°C and 37°C. Given that the CAL1 activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer toxin.
Purification and characterization of aminopeptidase yspI from Schizosaccharomyces pombe
Authors: Arbesú Ma José , Valle Eulalia , Suárez‐Rendueles Paz
Published: May 2005

Aminopeptidase yspI was purified to apparent homogeneity from the fission yeast Schizosaccharomyces pombe. The molecular mass of the native enzyme was estimated to be 184 kDa by gel filtration chromatography. A value of 92 kDa was calculated after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme is thus a dimer with two identical subunits. Optimum pH for cleavage of synthetic aminoacyl‐4‐nitroanilides is 7·0. Mercury ions, EDTA and chloroquine were found to be potent inhibitors of aminopeptidase yspI activity. Substrate specificity studies indicate that the purified enzyme cleaves L‐lysine‐4‐nitroanilide with high efficiency.
Vectors for the inducible overexpression of glutathione S‐transferase fusion proteins in yeast
Authors: Mitchell David A. , Marshall Tricia K. , Deschenes Robert J.
Published: May 2005

A rapid and convenient method of protein purification involves creating a fusion protein with glutathione S‐transferase (GST) (Smith and Johnson, Gene 67, 31–40, 1988). In this report, we describe two vectors for the conditional expression of GST fusions in Saccharomyces cerevisiae. The parent plasmid is based on a high‐copy, galactose‐inducible shuttle vector previously described (Baldari et al., EMBO J. 6, 229–243, 1987). We have demonstrated the use of this system by creating fusions between GST and the yeast RAS2 gene. GST‐Ras2 fusion proteins undergo the post‐translational modifications required for Ras2p to become membrane localized. These vectors provide a useful system for the expression an dpurification of eukaryotic proteins requiring post‐translational modification.
Regulation of glycolytic enzymes and the crabtree effect in galactose‐limited continuous cultures of Saccharomyces cerevisiae
Authors: Sierkstra Laurens N. , Nouwen Nico P. , Verbakel John M. A. , Verrips C. Theo
Published: May 2005

In order to determine whether the changes in the activities and mRNA levels of enzymes involved in intermediary carbon metabolism previously observed in glucose‐limited continuous cultures (Sierkstra et al., 1992a) were glucose specific, we have analysed their regulation in a galactose‐limited continuous culture of Saccharomyces cerevisiae. The Vmax of the galactose uptake system was shown to be dilution rate (D) dependent, comparable with the high‐affinity glucose uptake. The maximum uptake was observed at D 0·2 h−1 (0·25 mmol min−1 per g) and the minimum uptake (0·1 mmol min−1 per g) at D 0·05 h−1 and 0·3 h−1. The aerobic fermentation of galactose occurred at D 0·275–0·3 h−1 which is identical to the results obtained in glucose‐limited continuous cultures of this strain. Because galactose is not a repressing carbon source, this demonstrates that the Crabtree effect is not mediated by, or in any way related to glucose repression. Moreover, invertase and hexokinase I mRNA levels (both subject to glucose repression at the transcriptional level) were present when the yeast produced ethanol in galactose‐ and glucose‐limited continuous cultures. In glucose‐limited continuous cultures a decrease in alcohol dehydrogenase (I and II) mRNA levels and activity and phosphoglucomutase activity was observed with increasing dilution rates. In addition, at D 0·3 h−1, when the yeast produced ethanol, glucose‐6‐phosphate dehydrogenase and pyruvate decarboxylase were induced and a decrease in respiration was observed. The fact that the same changes were seen in a galactose‐limited culture and that invertase and hexokinase I mRNA levels were present demonstrates that glucose is not specifically involved in the regulation of these enzymes. The changes observed are therefore caused by the growth rate of the organism, the glycolytic flux (e.g. alcohol dehydrogenase) or the overflow metabolism when the yeast produces ethanol (e.g. pyruvate decarboxylase).
A 12·8 kb segment, on the right arm of chromosome II from Saccharomyces cerevisiae including part of the DUR1, 2 gene, contains five putative new genes
Authors: Bussereau Françoise , Mallet Laurent , Jacquet Michel , Gaillon Laurent
Published: May 2005

A 12 820 bp fragment from the right arm of chromosome II of Saccharomyces cerevisiae was sequenced and analysed. This fragment contains six non‐overlapping long open reading frames (ORFs) designated from the centromere‐ to the telomere‐proximal ends as: YBR1441, 1443, 1444, 1445, 1446 and 1448. YBR1441 encodes a polypeptide of 845 amino acids which shares a long consensus domain with products of S. cerevisiae MCM2, MCM3, CDC46 and Schizosaccharomyces pombe cdc21+ genes. These genes are involved in DNA replication. YBR1445 encodes a polypeptide of 404 amino acids which has strong similarity with the S. cerevisiae KRE2/MNT1, YUR1, KTR1 gene products. The KRE2/MNT1 protein is an α‐1,2‐mannosyltransferase. The product of YBR1444, which encodes a protein of 375 amino acids, presents a lipase signature sequence and a peroxisomal targeting signal. YBR1448, whose sequence extends further on the telomere‐proximal end of the fragment, is identical to the 3′ end of the DUR1,2 gene encoding urea amidolyase. The two ORFs, YBR1443 and YBR1446, exhibit no significant similarity with any known gene.
Analysis of the inducer‐responsive CAR1 upstream activation sequence (UASI) and the factors required for its operation
Authors: Kovari Ladislau Z. , Park Heui‐Dong , Kovari Iulia A. , Cooper Terrance G. , Fourie Marlene , van Vuuren Hendrik J. J.
Published: May 2005

Induced production of arginase (CAR1) enzyme activity and steady‐state CAR1 mRNA in Saccharomyces cerevisiae requires wild‐type ARG80/ARGRI and ARG81/ARGRII gene products. We demonstrate here that these gene products, along with that of the MCM1 gene, are required for the inducer‐dependent UASI‐A, UASI‐B and UASI‐C elements to function but they are not required for operation of inducer‐independent CAR1 UASC1 or UASC2M. Through the use of single and multiple point mutations, the CAR1 UASI‐B and UASI‐C elements were demonstrated to be at least 23 bp in length. Moreover, simultaneous mutation of both ends of an elements gave stronger phenotypes than mutations at either end. The center of the element was more sensitive to mutation than were the ends.
Sequence and function analysis of a 4·3 kb fragment of Saccharomyces cerevisiae chromosome II including three open reading frames
Authors: Schaaff‐Gerstenschläger Ine , Baur Axel , Boles Eckhard , Zimmermann Friedrich K.
Published: May 2005

The nucleotide sequence of a fragment of 4337 base pairs of Saccharomyces cerevisiae chromosome II has been determined. The sequence contains three open reading frames, one of them being incomplete. Deletion analysis showed that YBR12.31 is essential for yeast growth, while deletion mutants of YBR12.32 and YBR12.33 are viable. YBR12.33 is identical to SMY2, isolated as a suppressor of a myo2 mutant (Lillie, S.H. and Brown, S.S., unpublished, EMBL M90654).
Two distinct yeast proteins are related to the mammalian ribosomal polypeptide L7
Authors: Lalo Dominique , Mariotte Sylvie , Thuriaux Pierre
Published: May 2005

The RLP7 gene of Saccharomyces cerevisiae was cloned, sequenced and localized to the right arm of chromosome XIV, close to the centromere. It encodes a predicted polypeptide (RLP7p) of 322 amino acids, with a calculated molecular mass of 36 kDa and an isoelectric point of 9·6. Putative open reading frames very similar to RLP7 are present in two other yeasts, Kluyveromyces lactis and Candida utilis. The RLP7p gene product has significant sequence similarity to the S. cerevisiae YL8 polypeptide of the large ribosomal subunit (Mizuta et al., 1992), itself homologous to the L7 subunit of mammalian ribosomes. However, RLP7p and YL8 do not functionally replace each other, since an rlp7‐Δ::HIS3 strain is completely inviable. Judging from its predicted mass, isoelectric point and amino acid sequence, RLP7p does not correspond to any ribosomal component biochemically identified so far in S. cerevisiae, and also differs from all known ribosomal proteins by the low codon usage bias of its gene.