bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations
Authors: Hodgkins Martin , Sudbery Peter , Mead David , Ballance D. James , Goodey Andrew
Published: May 2005

The glucose oxidase gene (god) from Aspergillus niger was expressed in Hansenula polymorpha using the methanol oxidase promoter and transcription termination region and the MF‐α leader sequence from Saccharomyces cerevisiae to direct secretion. The expression cassette was cloned into the S. cerevisiae vector YEp13 and used to transform H. polymorpha strain A16. In the initial transformants plasmid replication was unstable, but was stabilized by a growth regime consisting of alternating cycles of selective and non‐selective growth. The stabilized strain was grown to high cell density by fed‐batch fermentation. Upon induction of the MOX promoter, glucose oxidase synthesis was initiated. At the end of the fermentation, the culture density was 76 g dry weight/1 and 108 IU/ml (0·5 g/l or 0·65% dry weight) glucose oxidase was found in the culture medium; a further 86 IU/ml (0·43 g/l or 0·56% dry weight) was recovered from the cell lysate. A plate assay was used to monitor glucose oxidase levels in individual colonies. This was then used to isolate mutants which showed abnormal regulation of god expression or which showed an altered pattern of secretion. One mutant, which showed increased production of glucose oxidase, was grown to high cell density by fed‐batch fermentation (100·6 g/l) and produced 445 IU/ml (2·25 g/l or 2·2% dry weight) extracellularly and 76 IU/ml (0·38 g/l or 0·4% dry weight) intracellularly. The mutant thus not only increased total production but exported 83% of the total enzyme made compared to 55% in the parent strain.
DNA sequence analysis of the YCN2 region of chromosome XI in Saccharomyces cerevisiae
Authors: Chéret Geneviève , Sor Frédéric , Mattheakis Larry C.
Published: May 2005

A 6·8 kbp DNA fragment localized to the left arm of chromosome XI from Saccharomyces cerevisiae was sequenced and analysed (EMBL accession no. X69765). Two genes involved in protein phosphatase activity were identified: YCN2 and an open reading frame encoding a protein that shares 46% amino acid identity with the sds22+ protein from Schizosaccharomyces pombe. A comparison of the genomic YCN2 sequence with the published cDNA sequence suggests the presence of an intron near the 5′ end of the gene. Further sequence analysis suggests the presence of three additional genes near YCN2: a mitochondrial acyl‐carrier protein, a gene encoding a putative hydrophobic protein, and a new gene coding for a tRNALeu (UAA) isoacceptor located near a delta sequence.
The In Vitro permeability of yeast peroxisomal membranes is caused by a 31 kDa integral membrane protein
Authors: Sulter G. J. , Harder W. , Verheyden K. , Mannaerts G. , Veenhuis M.
Published: May 2005

A major 31 kDa integral peroxisomal membrane protein (PMP31) of Hansenula polymorpha was purified to homogeneity from isolated peroxisomal membranes by FPLC after solubilization by Triton X‐100. Biochemical analysis indicated that this protein, which showed cross‐reactivity with antibodies against the 31 kDa porin of the mitochondrial outer membrane of Saccharomyces cerevisiae, had pore‐forming properties. Firstly, proteoliposomes composed of asolectin and purified PMP31 showed selective permeability, determined as the [14C]sucrose/[3H]dextran leakage ratios. Furthermore, the generation of a ΔΨ by potassium diffusion gradients was negatively affected by the presence of PMP31 in asolectin liposomes. A similar effect was observed in proteoliposomes containing purified cytochrome c oxidase as a ΔΨ generating system. Control experiments confirmed that the observed leakage is significant and introduced by the incorporation of PMP31 protein. Selective sucrose leakage was abolished in samples pretreated with glutaraldehyde; an identical effect of glutaraldehyde was, however, not observed for the membrane potential measurements.
Vector YFRp1 allows transformant selection in Saccharomyces cerevisiae via resistance to formaldehyde
Authors: Wehner Eugen P. , Brendel Martin
Published: May 2005

Formaldehyde (FA), a chemical with low toxic potential, is used as sole selective agent for transformation in the yeast Saccharomyces cerevisiae. Neither stable auxotrophic markers in recipient cells nor defined synthetic media are needed when multicopy vector YFRp1, containing the yeast SFA gene, is employed for yeast transformation. The SFA gene gives stability to the vector and its yeast (and other) passenger genes when transformants are propagated in complex media supplemented with 3–5 mM‐FA. Use of inexpensive FA and non‐synthetic, undefined media will lower the cost of yeast transformant propagation considerably and thus make feasible large‐volume industrial application of transformants containing YFRp1 derivatives.
CLG1, a new cyclin‐like gene of Saccharomyces cerevisiae
Authors: Matsumoto Yutaka , Wickner Reed B.
Published: May 2005

A region of chromosome VII adjacent to SKI8 has a 453 amino acid open reading frame whose sequence has significant similarity to that of HCS26, a G1 cyclin. A disruption mutation of this open reading frame has no apparent phenotype under the conditions tested. ORFD, an open reading frame adjacent to the CDC48 gene, is even more similar to HCS26.
Transcriptional regulation by glucose of the yeast PMA1 gene encoding the plasma membrane H+‐ATPase
Authors: Rao Rajini , Drummond‐Barbosa Daniela , Slayman Carolyn W.
Published: May 2005

The yeast plasma membrane H+‐ATPase generates a membrane electrochemical gradient which is required for the secondary uptake of nutrients. Although the ATPase has previously been shown to be post‐translationally regulated in response to the availability of glucose, there has been no evidence to date for transcriptional regulation of the ATPase gene (PMA1). In this work, we have examined the pool of newly synthesized ATPase that accumulates in secretory vesicles en route to the cell surface in the temperature‐sensitive secretory mutant sec6‐4, and have observed changes in the level of ATPase polypeptide as a function of the glucose concentration in the growth medium. In parallel, there were rapid and reversible changes in the levels of ATPase mRNA. Finally, when cells were grown on a variety of carbon sources, the amount of ATPase polypeptide was proportional to the specific growth rate, suggesting that PMA1 expression is adjusted according to the metabolic state of the cell. These results complement the findings of Capieaux et al. (Capieaux, E., Vignais, M.‐L., Sentenac, A. and Goffeau, A. (1989). J. Biol. Chem. 264, 7437–7446), who show that the transcriptional factor TUF/RAP1 binds to upstream activating sequences in the PMA1 gene. Taken together, the results suggest a model in which transcriptional regulation of the ATPase gene by glucose is mediated by TUF/RAP1.
Synonymous codon usage in Kluyveromyces lactis
Authors: Lloyd Andrew T. , Sharp Paul M.
Published: May 2005

The nature and variation of synonymous codon usage in 47 open reading frames from Kluyveromyces lactis have been investigated. Using multivariate statistical analysis, a single major trend among K. lactis genes was identified that differentiates among genes by expression level: highly expressed genes have high codon usage bias, while genes of low expression level have low bias. A relatively minor secondary trend differentiates among genes according to G+C content at silent sites. In these respects, K. lactis is similar to both Saccharomyces cerevisiae and Candida albicans, and the same ‘optimal’ codons appear to be selected in highly expressed genes in all three species. In addition, silent sites in K. lactis and S. cerevisiae have similar G+C contents, but in C. albicans genes they are more A+T‐rich. Thus, in all essential features, codon usage in K. lactis is very similar to that in S. cerevisiae, even though silent sites in genes compared between these two species have undergone sufficient mutation to be saturated with changes. We conclude that the factors influencing overall codon usage, namely mutational biases and the abundances of particular tRNAs, have not diverged between the two species. Nevertheless, in a few cases, codon usage differs between homologous genes from K. lactis and S. cerevisae. The strength of codon usage bias in cytochrome c genes differs considerably, presumably because of different expression patterns in the two species. Two other, linked, genes have very different G+C content at silent sites in the two species, which may be a reflection of their chromosomal locations. Correspondence analysis was used to identify two open reading frames with highly atypical codon usage that are probably not genes.
The DNA sequence analysis of the HAP4–LAP4 region on chromosome XI of Saccharomyces cerevisiae suggests the presence of a second aspartate aminotransferase gene in yeast
Authors: Chéret Geneviève , Pallier Chantal , Valens Michèle , Daignan‐Fornier Bertrand , Fukuhara Hiroshi , Bolotin‐Fukuhara Monique , Sor Frédéric
Published: May 2005

The nucleotide sequence of a 19 000 base pair region from the left arm of chromosome XI of Saccharomyces cerevisiae has been determined and analysed. It covers the HAP4–GFA1–LAP4 loci already described. As expected HAP4, GFA1 and LAP4 genes have been found and six new open reading frames (ORFs) with a coding capacity of more than 100 amino acid residues have been identified. One of them (YKL461) shows a high degree of identity with an aspartate aminotransferase gene. This raises the question of a second aspartate aminotransferase gene in yeast. A second ORF (YKL462) shows features compatible with a membranous localization. The other ORFs do not show a similarity with any known gene. A member of the highly repetitive ‘CAT’ DNA sequence is present.
Transport of hexoses in yeast. Re‐examination of the sugar phosphorylation hypothesis with a new experimental approach
Authors: Nevado Julián , Navarro MA Asunción , Heredia Claudio F.
Published: May 2005

The constitutive transport of hexoses in yeast has been re‐examined with a new radioactive experimental approach devised to distinguish between association or independence of the transport step with phosphorylation of the sugar substrate. The approach takes advantage of the fact that the label of [2‐3H]mannose disappears once it has been phosphorylated by the yeast, due to its conversion to fructose‐6‐phosphate. Our results with wild‐type yeast and this fermentable sugar support the view that the transport of hexoses in yeast does not involve phosphorylation of the substrate. Other features of the transport process have been examined using this experimental procedure and are also reported.
A homologous cell‐free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast
Authors: Brennwald Patrick , Wise Jo Ann
Published: May 2005

We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead‐beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER‐targeted proteins, pre‐acid phosphatase from S. pombe and prepro‐α‐factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane‐enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A–Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro‐α‐factor can be post‐translationally imported into the fission yeast ER, while S. pombe pre‐acid phosphatase crosses the membrane only by a co‐translational mechanism.
XI. Yeast sequencing reports. Sequence analysis of a 10 kb fragment of yeast chromosome XI identifies the SMY1 locus and reveals sequences related to a pre‐mRNA splicing factor and vacuolar ATPase subunit C plus a number of unidentified open reading frames
Authors: James Carolyn M. , Gent Manda E. , Indge Keith J. , Oliver Stephen G.
Published: May 2005

We report the DNA sequence analysis of a region on the left arm of chromosome XI of Saccharomyces cerevisiae extending over 10 kb. The region contains five open reading frames (ORFs) of greater than 100 amino acids which do not show significant overlap with other ORFs. YKL408 contains a sequence with strong similarity to the RNA helicase pre‐mRNA splicing factors PRP2, PRP16 and PRP22 (Burgess et al., 1990; Company et al., 1991; Ruby et al., 1991). YKL409 corresponds to the gene SMY1, the sequence of which was previously reported by Lillie and Brown (1992). YKL410 is identical to ATPase subunit C (Beltran et al., 1992) except for an N‐terminal extension. YKL406 and YKL407 show no significant identity with any sequences in the databases searched. The sequence has been entered in the EMBL Data Library under Accession Number X75560.
Sequence comparison of the ARG4 chromosomal regions from the two related yeasts, Saccharomyces cerevisiae and Saccharomyces douglasii
Authors: Adjiri Adouda , Chanet Roland , Mezard Christine , Fabre Francis
Published: May 2005

A 3·6 kb DNA fragment from Saccharomyces douglasii, containing the ARG4 gene, has been cloned, sequenced and compared to the corresponding region from Saccharomyces cerevisiae. The organization of this region is identical in both yeasts. It contains besides the ARG4 gene, another complete open reading frame (ORF) (YSD83) and a third incomplete one (DED81). The ARG4 and the YSD83 coding regions differ from their S. cerevisiae homologs by 8.1% and 12·5%, respectively, of base substitutions. The encoded proteins have evolved differently: amino acid replacements are significantly less frequent in Arg4 (2·8%) than in Ysc83 (12·4%) and most of the changes in Arg4 are conservative, which is not the case for Ysc83. The non‐coding regions are less conserved, with small AT‐rich insertions/deletions and 20% base substitutions. However, the level of divergence is smaller in the aligned sequences of these regions than in silent sites of the ORFs, probably revealing a higher degree of constraints. The Gcn4 binding site and the region where meiotic double‐strand breaks occur, are fully conserved. The data confirm that these two yeasts are evolutionarily closely related and that comparisons of their sequences might reveal conserved protein and DNA domains not expected to be found in sequence comparisons between more diverged organisms.
Purification of yeast histones competent for nucleosome assembly in vitro
Authors: Fukuma Mariko , Hiraoka Yoshiki , Sakurai Hiroshi , Fukasawa Toshio
Published: May 2005

We have developed a procedure to purify nucleosomal assembly‐competent histones as a mixture of H2A, H2B, H3 and H4 from isolated nuclei of the yeast Saccharomyces cerevisiae with a purity of 70–80%. The mixture contained each of the histone subunits approximately at the equi‐molar ratio. Plasmid pBR322 DNA was assembled into nucleosomes with the purified yeast histones in the presence of nucleoplasmin from unfertilized eggs of the frog Xenopus laevis. The efficiency of assembly of yeast histones was comparable to that of core histones purified from HeLa cells. The length of DNA fragment wrapping around a core histone particle and the molar ratio of histone components in an assembled nucleosome particle were estimated to be 150 ± 10 bp long and H2A:H2B:H3:H4 = 1·0:0·9:0·9:1·0, respectively.
XIIXVI. Yeast sequencing reports. Mapping and sequencing of two yeast genes belonging to the ATP‐binding cassette superfamily
Authors: Dean Michael , Allikmets Rando , Gerrard Bernard , Stewart Claudia , Kistler Amy , Shafer Brenda , Michaelis Susan , Strathern Jeff
Published: May 2005

ATP‐binding cassette (ABC) transporters share significant sequence identity within their ATP‐binding domains. Degenerate oligonucleotides based on highly conserved portions of the ATP‐binding domain genes were used to clone portions of two members of the ABC gene superfamily from Saccharomyces cerevisiae DNA. These genes were designated MDL1 and MDL2 (for multidrug resistance‐like). Each MDL gene is predicted to encode a single set of transmembrane domains and a single ATP‐binding domain, thus the MDL gene products are ‘half‐molecule’ ABC proteins. The two genes were mapped to precise regions on chromosomes XII and XVI and show a considerable similarity to the mammalian P‐glycoprotein/multidrug resistance (MDR) and peptide transporter (TAP) genes. Preliminary analysis of null mutants constructed by gene replacement has indicated that the MDL genes are not essential for viability of yeast. The sequences have been deposited in the GenBank data library under Accession Numbers L16958 (Locus YSCBCSA) and L16959 (Locus YSCBCSB).
Genetic evidence for a functional interaction between Saccharomyces cerevisiae CDC24 and CDC42
Authors: Ziman Michael , Johnson Douglas I.
Published: May 2005

Cdc24p and Cdc42p are involved in the control of cell polarity during the Saccharomyces cerevisiae cell cycle. Cdc42p is a member of the Ras superfamily of GTPases and Cdc24p displays limited amino‐acid sequence similarity with the Dbl proto‐oncoprotein, which acts to stimulate guanine‐nucleotide exchange on human Cdc42p. We have performed several genetic experiments to test whether Cdc24p and Cdc42p interact within the cell. First, overexpression of Cdc24p suppressed the dominant‐negative cdc42D118A allele. Second, overexpression of wild‐type CDC24 and CDC42 genes together was a lethal event resulting in a morphological phenotype of large, round, unbudded cells, indicating a loss of cell polarity. Third, a cdc24ts cdc42ts double mutant exhibited a synthetic‐lethal phenotype at the semi‐permissive temperature of 30°C. These data suggest that Cdc24p and Cdc42p interact within the cell and that Cdc24p may be involved in the regulation of Cdc42p activity.
X–XI. Yeast mapping reports. The use of random‐breakage mapping to locate the genes APN1 and YUH1 in the Saccharomyces genome, and to determine gene order near the left end of chromosome XI
Authors: Game John , Bell Maren , Ramotar Dindial , Miller Harvey
Published: May 2005

We have used the previously described technique of random‐breakage mapping to locate the two yeast genes APN1 and YUH1. The APN1 locus is located ∼235 kb from the left telomere of chromosome XI, and shows weak (∼53 cM) genetic linkage to ura1. The YUH1 locus is located ∼140 kb from the right telomere of chromosome X, and genetically maps 3·6 cM distal to cdc11. In addition, we show by random‐breakage mapping that TRP3 is located ∼45 kb from the left telomere of chromosome XI, whereas FAS1 is ∼110 kb from the same telomere. This supports a gene order on the left distal portion of chromosome XI that agrees with other physical reports but is inverted with respect to Edition 11 of the published genetic map. This report confirms that random‐breakage mapping is a rapid and convenient method of locating cloned genes.
Transfer RNA profiling: A new method for the identification of pathogenic Candida species
Authors: Santos Manuel A. S. , El‐Adlouni Chakib , Cox Alison D. , Luz José M. , Keith Gerard , Tuite Mick F.
Published: May 2005

A new molecular taxonomic method applicable to the identification of medically important Candida species and other yeast species has been developed. It is based on the electrophoretic pattern of total tRNA samples (a ‘tRNA profile’) isolated from Candida species and generated using high‐resolution semi‐denaturing urea‐polyacrylamide gel electrophoresis and methylene blue staining. Species‐specific tRNA profiles for the species. C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii, C. glabrata and Pichia guilliermondii were obtained. Detailed studies with the major human pathogen of the Candida genus, C. albicans, demonstrated that the tRNA profile for a given species was both reproducible and strain‐independent; seven different C. albicans strains generated identical tRNA profiles. Minor strain‐specific heterogeneities in the tRNA profiles of C. guilliermondii and C. parapsilosis were detected, but in neither case did they significantly alter the species‐specific diagnostic tRNA profile. The potential of this method in clarifying taxonomic anomalies was demonstrated by the finding that Type I and Type II strains of C. stellatoidea generate very different tRNA profiles, with that of a Type II strain being identical to the C. albicans tRNA profile. This method offers a number of advantages over current electrophoretic karyotype methods for species identification, both within the Candida genus and with yeast species in general.
IV. Yeast sequencing reports. A Saccharomyces cerevisiae gene encoding a potential adenine phosphoribosyltransferase
Authors: Yuryev Anton , Corden Jeffry L.
Published: May 2005

The nucleotide sequence of a Saccharomyces cerevisiae gene encoding a potential adenine phosphoribosyltransferase (APRT) has been determined. The protein encoded by this gene shows a high degree of similarity with APRTs from a variety of other species. The S. cerevisiae gene, named APT2, has been mapped to chromosome IV. The sequence has been deposited in the GenBank data library under Accession Number L14434.
IV. XV. Yeast mapping reports. RPL44 and RPL44′, encoding acidic ribosomal phosphoproteins YP2α(l44) and YP1β(l44′), are adjacent to rig and STF1 on Saccharomyces cerevisiae chromosomes XV and IV respectively
Authors: Rodriguez‐Gabriel M. A. , Zambrano R. , Ballesta J. P. G.
Published: May 2005

The RPL44′ gene from Saccharomyces cerevisiae encoding the ribosomal protein YP1β(L44′) has been found to be linked to the STF1 gene, encoding a stabilizing factor of the F1F0‐ATPase inhibitor protein from mitochondria. Evidence of this linkage comes from results obtained from Northern hybridization using a DNA probe that contains a complementary region to the 5′ end of the mRNA of RPL44′. Similarly, a data bank search has shown that RPL44, encoding ribosomal protein YP2α(L44) is linked to the rig gene that encodes ribosomal protein S21.
Phosphoribosylpyrophosphate synthetase (PRS): A new gene family in Saccharomyces cerevisiae
Authors: Carter Andrew T. , Narbad Arjan , Pearson Bruce M. , Beck Karl‐Friedrich , Baum Bobby , Logghe Marc , Contreras Roland , Schweizer Michael
Published: May 2005

Saccharomyces cerevisiae contains at least four PRS genes, all of which have been cloned and sequenced. Each of the four derived amino acid sequences have more than 60% similarity to the corresponding polypeptides of man, rat, Escherichia coli and Salmonella typhimurium. The PRS1 gene maps on chromosome XI, PRS2 on chromosome V, PRS3 on chromosome VIII and PRS4 on chromosome II. One member of this gene family, PRS1, contains a region of non‐homology (NHR) shown by cDNA cloning and sequencing not to be an intron. The results presented here suggest that the presence of this NHR is not detrimental to the function of the gene. To date the possibility of protein splicing can be neither proven nor disputed. The sequences submitted to the EMBL data library are available under the following accession numbers: PRS1 (X70069), PRS2 (X74414) and PRS3 (X74415).
nmt2 of fission yeast: A second thiamine‐repressible gene co‐ordinately regulated with nmt1
Authors: Manetti Andrea G. O. , Rosetto Marco , Maundrell Kinsey G.
Published: May 2005

We previously described a screen for thiamine‐repressible genes in Schizosaccharomyces pombe and reported on one such gene, nmt1, required for thiamine biosynthesis. Here we describe a second gene, nmt2, recovered in the same screen. Disruption of nmt2 also resulted in thiamine auxotrophy, indicating a role for the nmt2 gene product in thiamine biosynthesis. Both genes are highly transcribed in minimal medium and repressed in medium containing thiamine, and nuclear ‘run‐on’ experiments confirm that expression in both cases is controlled by the rate of transcription initiation. The virtually identical kinetics of induction and repression suggest that the two genes are co‐ordinately regulated. Sequence comparison of the two promoters reveals a canonical TATA box, downstream of which is a perfectly conserved 11 bp element. Transcript mapping experiments show that transcription initiation of both genes is centred on this element.
X. Yeast sequencing reports. The sequence of a 36 kb segment on the left arm of yeast chromosome X identifies 24 open reading frames including NUC1, PRP21 (SPP91), CDC6, CRY2, the gene for S24, a homologue to the aconitase gene ACO1 and two homologues to chromosome III genes
Authors: Purnelle Bénédicte , Coster Françoise , Goffeau André
Published: May 2005

A 36 kb fragment from the left arm of chromosome X, located at about 50 kb from the telomere, was sequenced and analysed. The segment contains a new putative ARS, a new tRNA for threonine, remnants of a solo delta and 24 open reading frames (ORFs) numbered from J0310 to J0355. Six of them, NUC1, PRP21 (also called SPP91), CDC6, CRY2, the gene encoding the ribosomal protein S24 and the gene coding for a hypothetical protein of 599 amino acids, have been sequenced previously. Three ORFs show high homology to the yeast gene ACO1 encoding mitochondrial aconitase and to the chromosome III genes YCR34W and YCR37C of unknown function. Three other ORFs show lower but significant homology: a first one to UNP, a gene related to the tre‐2 oncogene from mouse and to the gene coding for the yeast deubiquitinating enzyme DOA2; a second one to SLY41, a suppressor of the functional loss of YPT1 and a third one to the gene encoding the proline utilization activator PUT3.

The complete nucleotide sequence of 36 016 bp was submitted to the EMBL database (accession number X77688).
Detection of polygalacturonase, pectin‐lyase and pectin‐esterase activities in a Saccharomyces cerevisiae strain
Authors: Gainvors A. , Frézier V. , Lemaresquier H. , Lequart C. , Aigle M. , Belarbi A.
Published: May 2005

The catalytic capacity of several excreted pectinolytic enzymes obtained from various yeast strains was examined using in vivo and biochemical techniques. Of the 33 yeast strains studied, 30 were isolated from champagne wine during alcoholic fermentation. Only one yeast strain was found to excrete pectinolytic enzymes and was identified as Saccharomyces cerevisiae and designated SCPP. Pulsed‐field gel electrophoresis and the polymerase chain reaction technique were used to characterize further this specific strain. Three types of pectinolytic enzymes were found to be excreted by SCPP: polygalacturonase, pectin‐lyase and pectin‐esterase. These enzymes allow pectin hydrolysis during cell growth.
XIV. Yeast sequencing reports. Twelve open reading frames revealed in the 23·6 kb segment flanking the centromere on the Saccharomyces cerevisiae chromosome XIV right arm
Authors: Verhasselt Peter , Aert Rita , Voet Marleen , Volckaert Guido
Published: May 2005

The nucleotide sequence of 23·6 kb of the right arm of chromosome XIV is described, starting from the centromeric region. Both strands were sequenced with an average redundancy of 4·87 per base pair. The overall G+C content is 38·8% (42·5% for putative coding regions versus 29·4% for non‐coding regions). Twelve open reading frames (ORFs) greater than 100 amino acids were detected. Codon frequencies of the twelve ORFs agree with codon usage in Saccharomyces cerevisiae and all show the characteristics of low level expressed genes. Five ORFs (N2019, N2029, N2031, N2048 and N2050) are encoded by previously sequenced genes (the mitochondrial citrate synthase gene, FUN34, RPC34, PRP2 and URK1, respectively). ORF N2052 shows the characteristics of a transmembrane protein. Other elements in this region are a tRNAPro gene, a tRNAAsn gene, a τ34 and a truncated δ34 element. Nucleotide sequence comparison results in relocation of the SIS1 gene to the left arm of the chromosome as confirmed by colinearity analysis. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X77395.
Respiratory inhibitors affect incorporation of glucose into Saccharomyces cerevisiae cells, but not the activity of glucose transport
Authors: Walsh Michael C. , Smits Hans‐Peter , van Dam Karel
Published: May 2005

Incubation of starved galactose‐grown S. cerevisiae cells with cyanide reduced glucose uptake as measured over a 5‐s period. The Vmax for glucose uptake was decreased by over a factor of two but the apparent affinity for glucose doubled. When measured in the sub‐second time scale, however, there was no significant inhibition of glucose uptake, by cyanide, up to 200‐ms, clearly demonstrating that, in cyanide treated cells, glucose uptake was not linear for the first 5‐s.

After a 200‐ms exposure of untreated cells to radio‐labelled glucose, less than 10% of the intracellular label resided in soluble uncharged compounds. In cyanide‐treated cells up to 43% of the labelled compounds were uncharged, with a concurrent reduction of intracellular label residing in anionic compounds. The results suggest that, in the presence of 10 mM cyanide when respiration is inhibited, a reduction in the cellular ATP concentration causes a reduction in hexose‐kinase activity which results in an accumulation of internal free glucose, which in turn causes a reduction in net glucose transport.
Characterization of an active GST‐human Cdc2 fusion protein kinase expressed in the fission yeast Schizosaccharomyces pombe: A new approach to the study of cell cycle control proteins
Authors: Leroy Dorothée , Baldin Véronique , Ducommun Bernard
Published: May 2005

Characterization of cdk (cyclin dependent kinases) substrates and studies of their regulation require purified enzymatic complexes of cdc2‐related catalytic and cyclin regulatory subunits. We produced human Cdc2 kinase in the fission yeast Schizosaccharomyces pombe as a fusion protein with glutathione S‐transferase (GST). The GST‐human Cdc2p fusion protein was active in vivo since it rescued a temperature‐sensitive allele of cdc2. The fusion protein was purified using a one‐step chromatography procedure with glutathione–Sepharose and exhibited a catalytic activity in vitro. Yeast cyclin B and suc1 were found in association with GST‐Cdc2. A 17‐fold stimulation of GST‐Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with the S. pombe cellular extract prior to affinity purification. This indicates that cyclin concentration is limiting in this overexpression system. These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for biochemical studies.
The immunodetected yeast purine—cytosine permease is not N‐linked glycosylated, nor are glycosylation sequences required to have a functional permease
Authors: Rodriguez C. , Bloch J. C. , Chevallier M. R.
Published: May 2005

The purine‐cytosine permease (PCP) of the yeast Saccharomyces cerevisiae was detected by immunological methods. Using antibodies directed against synthetic peptides, whose sequences were derived from the primary structure of the PCP, immunoprecipitation of [35S]methionine‐labelled PCP was achieved either from cellular extracts or from in vitro translation mixtures. Non‐labelled PCP was also detected on Western blots of membrane proteins. Similar migration rates were observed for PCP originating both from immunoprecipitated cellular extracts and from in vitro translation mixtures. Hence, post‐translational processing, if any, only slightly affects the size of the protein. Also no evidence was found for N‐linked core‐glycosylation: identical migration rates were observed when immunoprecipitated PCP molecules were extracted from cells labelled for 10 min with [35S]methionine, pretreated or not with tunicamycin.

On the other hand, the suppresion of the two potential N‐linked glycosylation sequences in the DNA did not lead to inactivation of the transport activity, confirming that N‐linked glycosylation is not required for the permease activity.
An efficient method to isolate yeast genes causing overexpression‐mediated growth arrest
Authors: Espinet Carme , De La Torre Maria Angeles , Aldea Marti , Herrero Enrique
Published: May 2005

In order to characterize new yeast genes regulating cell proliferation, a number of overexpression‐sensitive clones have been isolated from a Saccharomyces cerevisiae cDNA library in a multicopy vector under the control of the GAL1 promoter, on the basis of growth arrest phenotype under galactose‐induction conditions. Thirteen of the independent clones isolated in this way correspond to previously known genes (predominantly coding for morphogenesis‐related proteins or for multifunctional transcriptional factors), while the remaining 11 independent clones represent new genes with unknown functions. The more stringent conditions employed in this screening compared with previous ones that also employed a dominant genetics approach to isolate overexpression‐sensitive genes has allowed us to extend the number of yeast genes that exhibit this phenotype. The effect of overexpression of MCM1 (whose product participates in the regulation of a number of apparently unrelated cellular functions) has been studied in more detail. Galactose‐induced overexpression of MCM1 leads to rapid growth arrest at the G1 or S cell cycle stages, with many morphologically‐abnormal cells. Several of the other clones also exhibit a G1 arrest terminal phenotype when overexpressed.
In vivo evidence for non‐universal usage of the codon CUG in Candida maltosa
Authors: Sugiyama Hiroki , Ohkuma Moriya , Masuda Yutaka , Park Sun‐Mee , Ohta Akinori , Takagi Masamichi
Published: May 2005

An alkane‐assimilating yeast Candida maltosa had been studied in order to establish systems suitable for biotransformation of hydrophobic compounds. However, functional expression of heterologous genes tested for this purpose had not been successful in several cases. On the other hand, it had been reported that the codon CUG, a universal leucine codon, is read as serine in C. cylindracea. The same altered codon usage had also been suggested by in vitro experiments in some Candida yeasts which are phylogenetically closely related to C. maltosa.

In this study we have shown that the failure in functional expression of a heterologous gene is due to the fact that the codon CUG is read as serine in C. maltosa. This conclusion was drawn from the following experimental results: (1) when a cytochrome P450 gene of C. maltosa containing a CTG codon was expressed in C. maltosa, the corresponding amino acid was found to be serine, and not leucine; (2) a tRNA gene with an almost identical structure to that of the tRNA SerCAG gene of C. albicans could be isolated from the genome of C. maltosa; (3) the Saccharomyces cerevisiae URA3 gene, which has one CTG codon, could not complement the ura3 mutation of C. maltosa as itself, but when the CTG codon was changed to another leucine codon, CTC, the mutated gene could complement the ura3 mutation.

The last result is the first example of succeeding in functional expression of a heterologous gene in Candida species having an altered codon usage by changing the CTG codon in the gene to another codon.

The nucleotide sequence datum reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the Accession Number D26074.
The sequence of a 13·5 kb DNA segment from the left arm of yeast chromosome XIV reveals MER1; RAP1; a new putative member of the DNA replication complex and a new putative serine/threonine phosphatase gene
Authors: Coster Francoise , Van Dyck Luc , Jonniaux Jean‐Luc , Purnelle Benedicte , Goffeau Andre
Published: May 2005

The nucleotide sequence of two adjacent ClaI fragments from the left arm of Saccharomyces cerevisiae chromosome XIV has been determined. Analysis of the 13,520 bp DNA segment reveals nine open reading frames (ORFs) longer than 300 bp. N1302 contains the consensus sequence for a phosphate‐binding loop common to ATP‐ and GTP‐binding proteins and a strictly conserved ‘SRC’ sequence of unknown function present in all accessory proteins of replicative polymerases. N1306 shares homologies with serine/threonine phosphatases. N1310 encodes RAP1 (TUF or SBF‐E), a transcription regulator. N1330 is the MER1 gene required for chromosome pairing and genetic recombination. Two ORFs show no homology with proteins in the databases and no particular features. N1311 is not likely to be expressed as it is located on the complementary strand of N1310. The sequence has been submitted to the EMBL data library under Accession Number X78898.
Construction of a complete genomic library of Saccharomyces cerevisiae and physical mapping of chromosome XI at 3·7 kb resolution
Authors: Thierry Agnès , Gaillon Laurent , Galibert Francis , Dujon Bernard
Published: May 2005

A consortium of European laboratories has been organized to systematically sequence the genome of Saccharomyces cerevisiae. As part of the BIOTECH program aimed at sequencing chromosomes XI and II, we have constructed a total genomic library of yeast strain FY1679 (a direct S288C derivative) into cosmid vectors pWE15 and pOU61cos. Primary clones from four independent libraries totalling 190 genome equivalents have been stored at −80°C.

A subset of 1939 independent clones (six genome equivalents) was hybridized using purified chromosomes XI and X as probes. A total of 147 chromosome XI‐specific cosmid clones was used to construct the physical map of that chromosome. Mapping methods included a combination of classical bottom‐up strategies (fingerprinting, hybridizations) and a novel top‐down strategy using I‐SceI chromosome fragmentation. The 147 cosmid clones form a unique contig covering the entire chromosome XI (666 kb) with the sole exceptions of the (C1−3A)n repeats of the telomeres. Colinearity of cosmid inserts with yeast DNA was directly verified. A complete EcoRI map of chromosome XI was deduced from partial overlaps of cosmids and used for the sequencing program. Comparison of this map with the genetic map shows unexpected divergences that have been solved by subsequent genetic analysis, yet underline the necessity of independent physical mapping in genome projects.
Recovery of gene function by gene duplication in Saccharomyces cerevisiae
Authors: Bach M. L. , Roelants F. , De Montigny J. , Huang M. , Potier S. , Souciet J. L.
Published: May 2005

A prototroph revertant (Rev9) selected from an ATCase− mutant of the URA2 gene containing three nonsense mutations was shown to contain two ATCase coding sequences. We cloned both ATCase coding areas to show that the duplicated locus (dl9) was the only functional one. Its size corresponded roughly to the second half of the URA2 wild‐type gene. Sequence analysis of the 5′ end of dl9 indicated that this duplicated sequence was inserted within the intergenic region close to the MRS3 gene and was transcribed from an unknown promoter divergently from the MRS3 gene. The event leading to the revertant strain Rev9 included a rearrangement that increased the size of chromosome X by about 60 kb. In agreement with such a rearrangement, recombination was undetectable in the vicinity of the locus dl9. Genetic mapping confirms that the MRS3 gene is 2 cM distal to the URA2 gene on the right arm of chromosome X.
Effects of phleomycin‐induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle
Authors: Belenguer Pascale , Oustrin Marie‐Louise , Tiraby Gérard , Ducommun Bernard
Published: May 2005

The effect of phleomycin, a bleomycin‐like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin‐induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2‐phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA‐damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.
UASNTR functioning in combination with other UAS elements underlies exceptional patterns of nitrogen regulation in Saccharomyces cerevisiae
Authors: Rai Rajendra , Daugherty Jon R. , Cooper Terrance G.
Published: May 2005

UASNTR, the UAS responsible for nitrogen catabolite repression‐sensitive transcriptional activation of many nitrogen catabolic genes in Saccharomyces cerevisiae, has been previously thought to operate only as a pair of closely related dodecanucleotide sites each containing the sequence GATAA at its core. Here we show that a single UASNTR site is also able to combine with another unrelated cis‐acting element to mediate transcription as well. In one instance the unrelated cis‐acting element was TTTGTTTAC situated upstream of GLN1, while in another the cis‐acting element was the one previously shown to bind the PUT3 protein. When a UASNTR site functions in combination with an unrelated site, the regulatory responses observed are a hybrid consisting of characteristics derived from both the UASNTR site and the unrelated site as well. These observations resolve several significant inconsistencies that have plagued studies focused on elucidation of the mechanisms involved in the global regulation of nitrogen catabolism.
Tandem integration of multiple ILV5 copies and elevated transcription in polyploid yeast
Authors: Mithieux Suzanne M. , Weiss Anthony S.
Published: May 2005

An industrial yeast strain was modified by introducing DNA into brewing yeast such that the derived cells contain only yeast DNA. Thus selectable markers and bacterial sequences are not present in the final strain, making this procedure attractive for the development of generally acceptable brewing yeast. Linear DNA containing the cloned ILV5 gene was introduced into lager yeast along with an unlinked circular bifunctional plasmid containing a dominant resistance marker. Resistant colonies were screened for site‐directed integration of the ILV5 DNA. Candidates were examined by several methods including Southern transfer and polymerase chain reaction. In this way, a strain WM56 was identified containing three tandem copies of ILV5. The amplified ILV5 region is stable during repeated subculturing in the absence of selective pressure. Correspondingly elevated levels of ILV5 transcript in strain WM56 compared to the control (i.e. non‐tandem) parental strain led to increased amounts of encoded acetohydroxyacid reductoisomerase as evidenced by significantly lower diacetyl production. WM56 appears to be identical to the parental strain judged by CHEF, total restriction digestion patterns, and probing, but differs in the ILV5 region of the chromosome. The method is generally applicable to other yeast strains, and if desired, is amenable to iterated cycles of integration to increase the number of copies.
Glucose metabolism, enzymic analysis and product formation in chemostat culture of Hanseniaspora uvarum
Authors: Venturin C. , Boze H. , Moulin G. , Galzy P.
Published: May 2005

The physiology of Hanseniaspora uvarum K5 was studied in glucose‐limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0·28 h−1, glucose was completely metabolized in biomass and CO2. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP‐dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO2 were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP‐dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl‐CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.
Isolation of the gene HEM4 encoding uroporphyrinogen III synthase in Saccharomyces cerevisiae
Authors: Amillet Jean‐Michel , Labbe‐Bois Rosine
Published: May 2005

We have isolated a genomic DNA fragment that complements the yeast temperature‐sensitive cyt mutation, causing respiratory deficiency and accumulation of porphyrins (Sugimura et al., 1966). Partial DNA sequencing of the complementing region and search for similarity in the DNA and protein databases revealed that (1) the gene had been previously isolated by complementation of the mutation ts2326 (Langgut et al., 1986; accession number X04694), and (2) it encodes a protein with 18–23% identity to uroporphyrinogen III synthases from different sources. This enzyme catalyses the fourth step in the heme biosynthetic pathway and we named its gene HEM4. A hem4Δ disruption mutation was constructed which had phenotypes identical to the cyt mutation. Biochemical analysis confirmed the absence of uroporphyrinogen III synthase activity in both hem4Δ and cyt mutant strains.
Sequence analysis of a 5·6 kb fragment of chromosome II from Saccharomyces cerevisiae reveals two new open reading frames next to CDC28
Authors: Baur Sabine , Becker Jûrgen , Li Ziyu , Niegemann Eckhard , Wehner Eugen , Wolter Ralf , Brendel Martin
Published: May 2005

The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28. Gene disruption reveals both putative genes as non‐essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Sur1 protein of S. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis‐secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product. The total sequence of the fragment has been submitted to the EMBL databank (accession number X80224).
Cloning and sequencing of the LEU2 homologue gene of Schwanniomyces occidentalis
Authors: Iserentant D. , Verachtert H.
Published: May 2005

A gene that complements the leu2 mutation of Saccharomyces cerevisiae has been cloned from Schwanniomyces occidentalis. The gene codes for a protein of 379 amino acids. As expected for a Schwanniomyces gene, it has a high AT content, which is also reflected in the codon usage. The sequence homology with other known leu2 complementing genes is low. The nucleotide sequence of the Schw. occidentalis LEU2 gene has been assigned the Accession Number X79823 SOLEU2 by EMBL.