bluuebunny/crossref_metadata_2025_split huggingface dataset:
The allantoinase (DAL1) gene of Saccharomyces cerevisiae
Authors: Buckholz Richard G. , Cooper Terrance G. 
Published: May 2005

AbstractThe allantoinase (DAL1) gene from Saccharomyces cerevisiae has been cloned, sequenced, and found to encode a 472 amino acid protein with a Mr of 52 028. DAL1 is expressed in an inducer‐independent manner in strain M970 (∑1278b genetic background) and modestly responds to mutation of the da180 locus. Expression was also sensitive to nitrogen catabolite repression (NCR). Correlated with these expression characteristics, the upstream region of DAL1 contained five copies of a sequence that is homolgous to the DAL UASNTR element previously shown to be required for transcriptional activation and NCR sensitivity of the DAL5 and DAL7 genes. Missing from the DAL1 5′ flanking region were any sequences with significant homology to the DAL7 UIS element required for response to inducer. These observations further support the roles of UASNTR and DAL7 UIS in the regulation of allantoin pathway gene expression.
Molecular cloning of CIF1, a yeast gene necessary for growth on glucose
Authors: González M. Isabel , Blázquez Miguel A. , Gancedo Carlos , Stucka Rolf , Feldmann Horst 
Published: May 2005

AbstractThe cif1 mutation of Saccharomyces cerevisia (Navon et al., Biochemistry 18, 4487–4499, 1979) causes inability to grow on glucose and absence of catabolite inactivation. We have cloned the CIF1 gene by complementation of funcion and licated it in a 2·75 kb SphI‐BstEII fragment situated at ca. 18 kb centomere distal of LYS2 and ca. 80 kb centromere proximal of TYRI on chromosome II. Southern analysis demostrated that CIF1 is present in a single copy in the yeast genome. Northern analysis revealed that the corresponding mRNA of 1·8 kb is more abundant in cells grown on galactose than in those grown on glucose. A protein of ca. 54 kDa was predicted from the open reading frame in the sequenced fragment. In strains carrying the cif1 mutation the intracellular concentration of ATP decreased immediately after addition of glucose while the intracellular concentration of cAMP did not increse. cAMP concentration increases in response to galactose or 2,4‐dinitrophenol. Disruption of BCY1 or overexpression of CDC25 in a cif1/, background did not restore growth on glucose, suggesting that the absence of cAMP signal is not primary cause of lack of growth on glucose. Complementation tests showed that cif1 is not allelic to fdp1 although the two genes seem to be functionally related.
Efficient secretion in yeast based on fragments from K1 killer preprotoxin
Authors: Cartwright Charles P. , Zhu Yun‐Song , Tipper Donald J. 
Published: May 2005

AbstractThe α and β components of the secreted K1 killer toxin of Saccharaomyces cerevisiae are derived from residues 45–147 and 234–316, respectively, of the 316 residue prepotoxin (ppTox). The β N‐terminus is produced by Kex2 cleavage after Lys Arg233, when β1a(the mature sequence of β‐lactamase)is fused at this site and the fusion is expressed form the PGK promoter in pDT17, a multicopy plasmid, unexpectedly modest levels of βla secretion resulted. Over‐expression of Kex2 failed to increase βla secretion while a kex2‐null mutation reduced secretion by 98%. βla secretion in a Kex+ strain was not enhanced by inactivation of the a toxin component or by deletion of most of its central hydrophobic segments. However SP‐βla, produced by deletion of ppTox residues 35–176, expressed 10‐fold higher βla activity and the precursor was not secreted with similar efficiency in a kex − 2 null strain. Fusions of βla to ppTox at Ala34 or Ala46 also led to efficient secretion in both KEX2 and kex − 2‐null strains. Since these βla fusions differ only in segments well downstream of the signal peptide and all had similar transcript levels, the efficiency of βla secretion is apparently determined by the efficiency with efficiency with which these fusions are translocated to the Golgi compartment where Kex2 is active. Efficiency is high for the shorter fusions but is 10% or less for the longer fusions; even this fraction is apparently diverted to the vacuole if not cleaved by Kex2. SP‐βla was athe most efficient construct tested; secreted βla reacahed 4% of total cell protein, modestly exceeding levels produced by fusion to the MFα1‐encoded preproα‐factor, suggesting potential for the production of foreign proteins in yeast.
Control of peroxisome proliferation in Saccharomyces cerevisiae by ADR1, SNF1 (CAT1, CCR1) and SNF4 (CAT3)
Authors: Simon Manuel , Binder Maximilian , Adam Gerhard , Hartig Andreas , Ruis Helmut 
Published: May 2005

AbstractThe Saccharomyces cerevisiae ADR1 gene has recently been demonstrated to control transcription of several genes encoding peroxisomal proteins or proteins necessary for peroxisome formation. Therefore, the effect of two other genes (SNF1 (CAT1, CCR1) and SNF4 (CAT3)) known to control derepression of glucose‐repressible genes was studied. Levels of transcripts of genes encoding catalase A, fatty acid β‐oxidation enzymes and of the PAS1 gene are reduced in snf1 and snf4 mutants of ethanol as well as on oleic acid medium. By immunogold labelling with an antibody directed against peroxisomal thiolase, clusters of peroxisomes were detected in wild‐types cells, whereas smaller single peroxisomes were observed in adr1 mutant cells. Results of immunofluorescence experiments are consistent with these observations. No peroxisomes were detected in snf1 and snf4 mutants by immunogold labelling as well as by imunofluorescence.
Sequence of a segment of yeast chromosome XI identifies a new mitochondrial carrier,a new member of the G protein family, and a protein with the PAAKK motif of the H1 histones
Authors: Colleaux Laurence , Richard Guy‐Franck , Thierry Agnès , Dujon Bernard 
Published: May 2005

AbstractWe have entirely sequaenced an 8.3 kb segment localized on the left arm of chromosome XI of Saccharomyces cerevisiae. Five new open reading frames have been uncovered. One of them encodes a new mitochondrial carrier protein which is dispensable for growth on glycerol medium. Another could be a new member of the G protein family. A third possesses the PAAKK motif common to H1 histones.
Genetic analysis of maintenance and expression of L and M double‐stranded RNAs from yeast killer virus K28
Authors: Schmitt Manfred J. , Tipper Donald J. 
Published: May 2005

AbstractThe killer phenotype expressed by Saccharomyces cerevisiae strain 28 differs fron that of the more extensively studied K1 and K2 killers with respect to immunity, mode of toxin action and cell wall primary toxin receptor. We previosly demonstrated that the M28 and L28 dsRNAs found in strain 28 are present in virus‐like particles (VLPs) and that transfection with these VLPs is sufficient to confer the complete K28 phenotype on a dsRNA‐free recipient cell. We also demonstrated that L28, like the L‐A‐H species in K1 killers, has [HOK] activity required for maintenance of M1‐dsRNA, and predicted that M28 would share with M1 dependence on L‐A for replication. We now confirm this prediction by genetic and biochemical analysis of the effects of representative mak, ski and mkt mutations on M28 maintenance, demonstrating that M28 replication resebles M1 in all respects. We also show that L28 is an L‐A‐H species lacking [B] activity, and that M28 excludes both M1 and M2 from the same cytoplasm. Stable coexpression of K28 phenotype from M28 and of K1 phenotype from an M1‐cDNA clone was demonstrated. Exclusion, therefore, acts at the level of dsRNA replication, presumably reflecting competition for the L‐A‐H encoded capsid and cap‐pol fusion protein, rather than reflecting incompatibility of toxin or immunity expression. Finally, we show that expression of active K28 toxin, bu t not of K28 immunity, requires the Kex2 endoprotease.
The complete sequence of a 10.8 kb segment distal of SUF2 on the right arm of chromosome III from Saccharomyces cerevisiae reveals seven open reading frames including the RVS161, ADP1 and PGK genes
Authors: Skala Jacek , Purnelle Bénedicte , Goffeau André 
Published: May 2005

AbstractWe have entirely sequenced a 10,835 bp segment of the right arm from chromosome III contained in the J11D and J11D‐K3B GF clones. The segment contains seven open reading frames longer than 100 amino acids. Three of them, RVS161 (Urdaci et al., 1990; Crouzet et al., 1991), ADP1 (Purnelle et al., 1991) and PGK1, (Hitzeman et al., 1982) have been described previously. YCR10C, encodes a putative membrane protein. YCR8W, (encoding a putative protein kinase) and YCR14c extend inside the D10H (Skala et al., 1991) and 62B5‐2D clones respectively. Four ARS elements previously reported by Palzkill et al., (1986) are located between RVS161 and YCR10C.
Factors affecting homologus overexpression of the Sacchromyces cerevisiae Lanosterol 14α‐demethylase gene
Authors: Weber Jakob M. , Ponti Charly G. , Käppeli Othmar , Reiser Jakob 
Published: May 2005

AbstractThe Saccharomyces cerevisiae Lanosterol 14α‐demethylase (14DM) gene was overxpressed in S. cerevisiae using Promoter sequences of the highly expressed S. cerevisiae glyceraldehyde‐3‐phosphate dehydrogenase TDH3 gene. To investigate factors affecting 14DM overproduction, the levels of 14DM‐specific specific RNAs, apoprotein, and heme protein, repectively, were determined and the 14DM‐specific RNA levels compared with the RNA levels originating from the enodogenous TDH gene(s). The quantitative measurements revealed that the 14DM steady‐state RNA levels reached were some three‐to five‐fold below the theoretically expected values. With a View towards futrher improving expression of the 14DM gene, the specing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment. Furthermore, the effects of the gene copy number on 14DM overproduction were investigated. From the analysis of the improved expression constructs five conclusions could be reached: (1) experssion from the native 14DM gene is comparable to the expression driven by the TDH3 promoter‐14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter‐14DM construct on single‐copy vectors is nearly as effcient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter‐14DM constructs; (4) the steady‐state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production. The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.
Analysis of the MSS51 region on chromosome XII of Saccharomyces cerevisiae
Authors: Simon Michael , Seta Flavio Della , Sor Frederic , Faye Gerard 
Published: May 2005

AbstractWe have localized gene MSS51 on chromosome XII of Saccharomyces cerevisiae between the RDN1 and CDC42 loci. ‘Head to head’ with MSS51 is another gene, QRI5, the function of which is unkown. However, the proximity of these genes, the structure of the intergenic region and the presence of an ABFl binding site right in the middle of this region suggest that the MSS51 and QRI5 expressions are submitted to a common regulatory process.
The complete sequence of a 6146 bp fragment of Saccharomyces cerevisiae chromosome III contains two new open reading frames
Authors: Wilson Cathal , Grisanti Paola , Frontal Laura 
Published: May 2005

AbstractAs part of the EEC project to sequence the entire chromosome III of Saccharomyces cerevisiae we have sequenced a total of 11,040 bp from near the right end of the chromosome. A new protein kinase gene was found at one extremity of the sequenced region (Wilson et al., 1992), while the previouslysequenced actin binding protein gene, ABPI, (Drubin et al., 1990) was found at the other extremity. We present here the sequence of the region between these two genes which has the potential to code for two new open reading frames (ORFs).
II. Yeast sequencing reports. An 11·4 kb DNA segment on the left arm of yeast chromosome II carries the carboxypeptidase Y sorting gene PEP1, as well as ACH1, FUS3 and a putative ars
Authors: Van Dyck Luc , Purnelle Bénédicte , Skala Jacek , Goffeau André 
Published: May 2005

AbstractWe report the nucleotide sequence of an 11.4 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome II. This sequence contains a typical structure of a functional ARS as well as five open reading frames (ORFs) longer than 300 bp. One is PEP1, a gene encoding a transmembrane protein of 1579 amino acids which transits through the secretory pathway and is involved in vacuolar protein sorting. Two genes were previously sequenced: ACH1 (Lee et al., 1990) and FUS3 (Elion et al., 1990), which encode an acetyl‐CoA hydrolase and a protein kinase involved in the cell division cycle, respectively. The last two ORFs localized on the complementary strand of ACH1 are not likely to be expressed.
Isolation and characterization of peroxisomal protein import (Pim−) mutants of Hansenula polymorpha
Authors: Waterham Hans R. , Titorenko Vladimir I. , Van Der Klei Ida J. , Harder Wim , Veenhuis Marten 
Published: May 2005

AbstractIn the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim− phenotype). Genetic analysis of back‐crossed mutants revealed five different complementation groups, which were designated PERI–PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim− phenotypes were determined by single recessive nuclear mutations.The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome‐borne enzymes in wild‐type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild‐type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.
Effect of sterol alterations on conjugation in Saccharomyces cerevisiae
Authors: Tomeo Michele E. , Fenner Gregeory , Tove Shirley R. , Parks Leo W. 
Published: May 2005

AbstractSterol auxotrophic strains of Saccharomyces cerevisiae were grown and allowed to conjugate on media supplemented with various sterols.The mating efficiency of the auxotrophs is perturbed by the relacement of the normal yeast sterol, ergsterol, with other sterols. After 4 h of mating, cells grown on ergosterol a 30‐fold higher productive mating efficiency than those cells grown in stigmasterol. Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non‐ergosterol sterols. Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol‐grown cells to undergo cytoplasmic fusion during conjugation. Many of the mated pairs remained adherent but Prezygotic even after 12 h of incubation. The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal budding.
The SCH9 protein kinase mRNA contains a long 5′ leader with a small open reading frame
Authors: Di Blasi Francesco , Carra Elena , De Vendittis Emmanuele , Masturzo Pietro , Burderi Emanuele , Lambrinoudaki Irene , Mirisola Mario G. , Seidita Gregorio , Fasano Ottavio 
Published: May 2005

AbstractThe SCH9 yeast gene, that was previously identified as a suppressor of cdc25 and ras1− ras2‐ts temperature‐sensitive mutants, encodes a putative protein kinase that positively regulates the progression of yeast cells through the G1 phase of the cell cycle. We have determined the structure of the SCH9 transcription unit, using primer extension and S1 mapping techniques. The corresponding mRNA included an unusually long 5′ region of more than 600 nucleotides preceding the major open reading frame (ORF). While the latter corresponded to a protein of 824 amino acids, an upstream open reading frame (uORF) within the 5′ leader could potentially encode a 54 amino acid peptide. To investigate the role of the AUGs within the uORF, we engineered chimaeric plasmid vectors in which SCH9 sequences including the promoter, the mRNA leader and the first 514 nucleotides of the major ORF were fused in‐frame with β‐galactosidase‐coding sequences. Upon introduction into yeast cells, the fusion protein was efficiently expressed. However, mutational disruption of the uORF using oligonucleotide‐directed mutagenesis did not affect the level of expression of the fusion protein. This indicates that regulatory mechanisms in Saccharomyces cerevisiae prevent upstream AUGs within the SCH9 mRNA leader sequence from influencing translation from downstream initiation codons.
Cloning and genetic analysis of the gene encoding a new protein kinase in Saccharomyces cerevisiae
Authors: Kambouris Nicholas G. , Burke Daniel J. , Creutz Carl E. 
Published: May 2005

AbstractWe have isolated a single gene from the yeast Saccharomyces cerevisiae encoding a potential 800 amino acid polypeptide of calculated Mr 90 098 Da. This protein consists of an N‐terminal region that shares significant homology with the catalytic domains of several serine‐ and threonine‐specific protein kinases, as well as a large, unique, C‐terminal domain of unknown function. Haploid disruption mutants are viable and do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients or osmotic strength. Due to the apparent structural similarity between this kinase and the protein products of the KIN1 and KIN2 genes, we have chosen to name this new gene KIN3.
Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation
Authors: Bordallo Javier , Suárez‐Rendueles Paz 
Published: May 2005

AbstractExpression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients. Enzyme production is sensitive to nitrogen catabolite repression; i.e. the presence of ammonium ions maintains expression of the gene at a low level. Transfer of ammonium–glucose pre‐grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells. Derepression of the gene by nitrogen limitation is cycloheximide‐insensitive. Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions. Non‐metabolizable sugar analogs (2‐deoxyglucose, 6‐methyl‐glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy‐dependent. Production of carboxypeptidase yscS also increases several‐fold when ammonium‐pregrown cells are transferred to media containing glucose and a non‐readily metabolizable nitrogen source such as proline, leucine, valine or leucyl‐glycine. Analysis of CPS1 expression in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23°C) and in heat‐shocked cells (38°C) shows that steady‐state levels of CPS1 mRNA are not controlled by a low cAMP level‐signalling pathway.
A new promoter‐probe vector for Saccharomyces cerevisiae using fungal glucoamylase cDNA as the reporter gene
Authors: Scorpione Regina Céli , De Camargo Solange Soares , Astolfi‐Filho Spartaco , Schenberg Ana Clara Guerrini 
Published: May 2005

AbstractA system is described for the selection of DNA sequences showing promoter activity in the yeast Saccharomyces cerevisiae using a heterologous reporter enzyme which is efficiently secreted by the yeast host. A multicopy shuttle plasmid of the YEp‐type was constructed so as to carry multiple unique cloning sites at the 5′ end of the Aspergillus awamori glucoamylase cDNA. Glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a DNA fragment from any source, provided it is endowed with promoter function in S. cerevisiae. As the glucoamylase signal‐peptide is functional in S. cerevisiae, the enzyme is efficiently secreted by the yeast transformants. This phenotype can be very easily detected on plate assays and accurately quantified by spectrophotometric analysis of the culture supernatant. Since S. cerevisiae naturally lacks amylolytic activity, any wild‐type strain can be used as a host in this system. To evaluate the system, a DNA pool of random fusions was created by ligating sau 3A digested S. cerevisiae genomic DNA to the BglII‐linearized vector. The resulting hybrid plasmids were transformed into S. cerevisiae and several transformants secreting glucoamylase to varying degrees were obtained.
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

AbstractThe 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

AbstractA 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.