bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic and carbon source regulation of phosphorylation of Sip1p, a Snf1p‐associated protein involved in carbon response in Saccharomyces cerevisiae
Authors: Long Roy M. , Hopper James E. 
Published: May 2005

AbstractThe SIP1 gene of Saccharomyces cerevisiae is a carbon‐catabolite‐specific negative regulator of GAL gene transcription and acts as a multicopy suppressor of growth defects associated with impaired Snf1p protein kinase activity. The Sip1 protein is known to undergo phosphorylation when associated in vitro with the Snf1 protein kinase. We have carried out in vivo studies of the genetic and carbon control of Sip1p phosphorylation. Metabolic labeling reveals phosphorylation of Sip1p under both carbon catabolite‐repressing and non‐repressing conditions and in both SNF1 wild‐type and snf1‐deletion cells. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis immunoblot assay, we detect apparent changes in Sip1p phosphorylation states in response to changes in carbon source. At least one dephosphorylation of Sip1p occurs with a shift from non‐repressing carbon source to repressing carbon source. The MIG1 gene, acting through SNF1‐dependent and SNF1‐independent pathways, is required for some Sip1p phosphorylations. REG1 appears to be required for at least one dephosphorylation of Sip1p, whereas SSN6 appears to be required for at least one phosphorylation of Sip1p. These results reveal new complexities in carbon response signaling, and may reflect the involvement of the Sip1 protein in the same complex as the Mig1 and Ssn6 proteins.
Cloning and heterologous expression of the Candida albicans gene PMI 1 encoding phosphomannose isomerase
Authors: Smith David J. , Proudfoot Amanda E. I. , Detiani Mariastella , Wells Timothy N. C. , Payton Mark A. 
Published: May 2005

AbstractUsing a DNA fragment derived from the Saccharomyces cerevisiae phosphomannose isomerase (PMI) structural gene as a probe against a random ordered array library of genomic DNA from the pathogenic fungus Candida albicans, we have cloned the C. albicans PMI 1 gene. This gene, which is unique in the C. albicans genome, can functionally complement PMI‐deficient mutants of both S. cerevisiae and Escherichia coli. The DNA sequence of the PMI 1 gene predicts a protein with 64·1% identity to PMI from S. cerevisiae. Sequential gene disruption of PMI 1 produces a strain with an auxotrophic requirement for D‐mannose. The heterologous expression of the PMI 1 gene at levels up to 45% of total cell protein in E. coli leads to partitioning of the enzyme between the soluble and particulate fractions. The protein produced in the soluble fraction is indistinguishable in kinetic properties from the material isolated from C. albicans cells. The nucleotide sequence data reported here will appear in the EMBL database under Accession Number X82024.
Plasmid reorganization during integrative transformation in Hansenula polymorpha
Authors: Bogdanova Aliona I. , Agaphonov Michael O. , Ter‐Avanesyan Michael D. 
Published: May 2005

AbstractDuring studies of integrative transformation in Hansenula polymorpha, it was found that transformants with plasmids possessing the LEU2 gene of H. polymorpha were frequently unstable and lost plasmids while growing on non‐selective medium. These transformants possessed reorganized plasmids capable of replication in H. polymorpha. Two such plasmids were isolated and characterized. It was shown that they contain additional DNA segments which were not present in the original plasmid used for transformation. Southern hybridization analysis carried out with labeled DNA probes derived from these segments showed that they consisted of H. polymorpha DNA. The hybridization patterns indicated that corresponding sequences were homologous to several chromosomal regions. These chromosomal DNA segments apparently carried H. polymorpha autonomous replicating sequences (HARS), since plasmids bearing them could transform H. polymorpha with high efficiency and were maintained in transformants in an autonomous state. Sequence analysis of one such captured chromosomal fragment revealed several eight‐ to ten‐base AT‐rich blocks similar to the presumed HARS sequence defined by Roggenkamp et al. (1986). Analogous reorganization was also observed with respect to integrative plasmids carrying the TRP3 and HIS3 genes of H. polymorpha and the ADE2 gene of Saccharomyces cerevisiae as selectable markers.
The upstream sequences of the HSP82 and HSC82 genes of Saccharomyces cerevisiae: Regulatory elements and nucleosome positioning motifs
Authors: Erkine Alexander M. , Szent‐Gyorgyi Christopher , Simmons Serena F. , Gross David S. 
Published: May 2005

AbstractWe present the upstream sequences of HSP82 and HSC82, two closely related, but differentially regulated, heat‐shock genes of Saccharomyces cerevisiae. Several dozen potential regulatory elements are identified within each upstream region; interestingly, only a few are conserved between the two genes. These include a consensus heat‐shock element, an upstream repressor element, and a consensus TATA element. A search for motifs known actively to position nucleosomes in vitro revealed that such sequences are three‐ to seven‐fold enriched within each promoter; a comparable enrichment is seen near the 3′ end of each transcription unit. Located ∼ 1100 bp upstream of HSC82 is an open reading frame (ORF) of 255 amino acids; ∼ 800 bp upstream of HSP82 is an ORF of 132 amino acids. The latter ORF contains several conserved ankyrin motifs and appears to be expressed under normal growth conditions. Finally, we show by clamped homogeneous electric field gel electrophoresis that the two genetic loci map to different chromosomes: HSP82 to chromosome XVI and HSC82 to chromosome XIII. The sequences have been deposited in the GenBank database under Accession Numbers U20323 and U20349.
Kluyveromyces lactis killer plasmid pGKL2: Molecular analysis of an essential gene, ORF5
Authors: Schaffrath Raffael , Meacock Peter A. 
Published: May 2005

AbstractThe ORF5 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a small neutral protein of 18 kDa of as yet unassigned function. Although this ORF is located between two larger ORFs, 4 and 6, which it overlaps, RNA analysis showed that it is transcribed monocistronically. One‐step gene disruption of ORF5, via in vivo homologous recombination between native plasmid k2 and a transfer vector employing the Saccharomyces cerevisiae LEU2 gene fused to the k2 UCS5 element, yielded Leu+ transformants at high frequencies. The transformants were found to carry a new recombinant form of k2 with ORF5 replaced by the LEU2 marker, termed rk2, in addition to the wild‐type plasmids k1 and k2. Northern analysis detected a plasmid‐dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart. Recombinant plasmid, rk2, was unable to displace native k2 during Leu+ selective growth; however rk2 was displaced by k2 during non‐selective growth. Thus, ORF5 appears to be an essential gene for plasmid integrity and/or maintenance. The ORF5 product was detected by over‐expression of an epitope‐tagged allele in the baculovirus system. Western analysis using a monoclonal antibody specific for the epitope tag identified a protein band with apparent molecular weight of 20 kDa, corresponding in size to the predicted product.
Sequence of a 9·8 kb segment of yeast chromosome II including the three genes of the MAL3 locus and three unidentified open reading frames
Authors: Feuermann M. , Charbonnel L. , De Montigny J. , Bloch J.‐C. , Potier S. , Souciet J.‐L. 
Published: May 2005

AbstractWe report the DNA sequence of a segment located on the right arm of chromosome II from Saccharomyces cerevisiae S288C near the subtelomeric sequences. The sequence was determined using a random cloning strategy followed by an oligonucleotide‐directed sequencing. The segment contains four non‐overlapping open reading frames (ORFs) YBR297w, YBR298c, YBR299w and YBR301c, and two overlapping ones (YBR300c and YBR300w). Three of them—YBR297w, YBR298c and YBR299w—are the MAL3R (transcriptional regulatory protein), MAL3T (maltose permease) and MAL3S (maltase) genes of the MAL3 locus previously localized. The three other ORFs are unidentified. Another MAL locus (MAL1) has been localized on chromosome VII. The Mal− phenotype of strain S288c cannot be explained by telomeric silencing. The sequences have been submitted to the EMBL data library under Accession Numbers Z36166; Z36167; Z36168; Z36169 and Z36171.
Analysis of a 32·8 kb segment of yeast chromosome IV reveals 21 open reading frames, including TPS2, PPH3, RAD55, SED1, PDC2, AFR1, SSS1, SLU7 and a tRNA for arginine
Authors: Coster Françoise , Jonniaux Jean‐Luc , Goffeau Andre 
Published: May 2005

AbstractWe report the nucleotide sequence of a 32·8 kb DNA segment from the right arm of Saccharomyces cerevisiae chromosome IV. The sequence contains 20 open reading frames (ORFs) longer than 300 bp as well as the 240 bp gene coding for the essential SSS1 secretory protein. Nine ORFs previously totally or partially sequenced (TPS2, PPH3, RAD55, SED1, PDC2, AFR1, SSS1, SLU7 and D4478) are presented, as well as the transmembrane protein D4405, the leucine zipper containing D4495 and a new tRNA for arginine. D4456 and D4461 are separated by a single in‐frame stop codon only. The other five ORFs show no particular features or significant homology. The sequence is recorded in EMBL database under Accession Number X82086.
Localization of the FAR3 gene: Genetic mapping and molecular cloning using a chromosome walk‐‘n’‐roll strategy
Authors: Horecka Joe 
Published: May 2005

AbstractFAR3 is a newly‐discovered yeast gene required specifically for pheromone‐mediated cell cycle arrest. I have used strains harboring the far3‐1 mutation to map the gene to the right arm of chromosome XIII, establishing the gene order CEN13‐LYS7‐MCM1‐FAR3. I cloned the FAR3 gene based on its genetic map position using a strategy that combined chromosome walking and a related technique termed ‘chromosome rolling’. In addition to the genetic and physical localization of FAR3, I present data that suggest corrections to the tentative map positions of VAN1 and ARG80.
Characteristics of spontaneous revertants in haploid yeast
Authors: Korogodina V. L. , Korogodin V. I. , Maiorova E. S. , Simonyan N. V. 
Published: May 2005

AbstractThe appearance dynamics of spontaneous revertants in adenine‐ or leucine‐auxotrophic haploid Saccharomyces cerevisiae strains has been studied using mathematical simulation methods. In the case of adenine auxotrophs an increase in the number of revertants with decreasing metabolite content is found to result mainly from increasing the rate of intragenic suppressor mutations. In the case of leucine auxotrophs revertants result from increasing the appearance of mutants formed at similar rates under different cultivation conditions. In the latter case the appearance of mutant colonies increases with decreasing size of colonies of the initial auxotrophic cells. The last can simulate so‐called adaptive mutagenesis. The heterogeneity of revertants appearing in different time periods is described.
Characterization of Na+/H+‐antiporter gene closely related to the salt‐tolerance of yeast Zygosaccharomyces rouxii
Authors: Watanabe Yasuo , Miwa Satoko , Tamai Youichi 
Published: May 2005

AbstractIn order to clarify the relationship between salt‐tolerance of Zygosaccharomyces rouxii and the function of Na+/H+‐antiporter, a gene was isolated from Z. rouxii which exhibited homology to the Na+/H+‐antiporter gene (sod2) from Schizosaccharomyces pombe. This newly isolated gene (Z‐SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by its Sz. pombe homologue. The predicted amino‐acid sequence of Z‐Sod2p was highly homologous to that of the Sz. pombe protein, but included an extra‐hydrophilic stretch in the C‐terminal region. The expression of Z‐SOD2 was constitutive and independent of NaCl‐shock. Z‐SOD2‐disruptants of Z. rouxii did not grow in media supplemented with 3 M‐NaCl, but grew well in the presence of 50% sorbitol, indicating that the function of Z‐SOD2 was closely related to the salt‐tolerance of Z. rouxii. Several genes are also compared and discussed in relation to the salt‐tolerance of Z. rouxii. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the following accession number: D43629.
Expression and secretion of antifreeze peptides in the yeast Saccharomyces cerevisiae
Authors: Driedonks R. A. , Toschka H. Y. , van Almkerk J. W. , Schäffers I. M. , Verbakel J. M. A. 
Published: May 2005

AbstractThe antifreeze peptide AFP6 from the polar fish Pseudopleuronectus americanus has been expressed in and secreted by the yeast Saccharomyces cerevisiae as a biologically active molecule. The gene for the 37 amino acid long peptide has been chemically synthesized using yeast preferred codons. Subsequently, the gene has been cloned into an episomal expression vector as well as in a multicopy integration vector, which is mitotically more stable. The expression is under the control of the inducible GAL7 promoter. The enzyme α‐galactosidase has been investigated as a carrier protein to facilitate expression and secretion of AFP. In order to reach increased expression levels, tandem repeats of the AFP gene (up to eight copies) have been cloned. In most cases the genes are efficiently expressed and the products secreted. The expression level amounts to approximately 100 mg/l in the culture medium. In a number of genetic constructs the genes are directly linked and expressed as AFP multimers. In other constructs linker regions have been inserted between the AFP gene copies, that allow the peptide to be processed by specific proteinases, either from the endogenous yeast proteolytic system or from a non‐yeast source. The latter requires a separate processing step after yeast cultivation to obtain mature AFP. In all these cases proteolytic processing is incomplete, generating a heterogeneous mixture of mature AFP, carrier and chimeric protein, and/or a mixture of AFP‐oligomers. The antifreeze activity has been demonstrated for such mixtures as well as for AFP multimers.
Sequence and functional analysis of a 7·2 kb DNA fragment containing four open reading frames located between RPB5 and CDC28 on the right arm of chromosome II
Authors: Rose Matthias , Kiesau Peter , Proft Markus , Entian Karl‐Dieter 
Published: May 2005

AbstractIn a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28. The fragment contains four open reading frames probably encoding proteins of 79·2 kDa (corresponding gene YBR156c), 12·1 kDa (YBR157c), 62·7 kDa (YBR158w) and 38·7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated. Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grr1 (Cat80) protein with respect to the leucine‐rich region. Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild‐type cells. The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17‐beta‐hydroxysterol dehydrogenase 3. Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild‐type strains. The DNA sequence was deposited at the EMBL data base with accession numbers Z36025 (YBR156c), Z36026 (YBR157c), Z36027 (YBR158w) and Z36028 (YBR159w).
Transcriptional regulation by an upstream repression sequence from the yeast enolase gene ENO1
Authors: Carmen Andrew A. , Brindle Paul K. , Park Chang Seo , Holland Michael J. 
Published: May 2005

AbstractThe activity of an upstream repression sequence (URS element) that mediates a 20‐fold repression of ENO1 expression in cells grown in a medium containing glucose was characterized. Sequences that are sufficient for orientation‐dependent ENO1 URS element activity were mapped between positions ‐241 and ‐126 relative to the ENO1 transcriptional initiation site. The ENO1 URS element repressed transcription of the yeast CYC1 gene when positioned between the CYC1 upstream activation sequences (UAS elements) and TATAAA boxes. The ENO1 URS element failed to repress transcription of the wild‐type yeast enolase gene ENO2; however, expression of an ENO2 gene lacking one of the ENO2 UAS elements was efficiently repressed by the ENO1 URS element, suggesting that the URS element interferes with the transcriptional activation by some, but not all, UAS elements. In contrast to the ENO1 gene, the ENO1 URS element repressed CYC1 and ENO2 expression in cells grown on glucose or glycerol plus lactate. Evidence is presented that the ENO1 URS element also functions during stationary growth phase.
Identification of peroxisomal membrane ghosts with an epitope‐tagged integral membrane protein in yeast mutants lacking peroxisomes
Authors: Purdue P. Edward , Lazarow Paul B. 
Published: May 2005

AbstractMany yeast peroxisome biogenesis mutants have been isolate in which peroxisomes appear to be completely absent. Introduction of a wild‐type copy of the defective gene causes the reappearance of peroxisomes, despite the fact that new peroxisomes are thought to form only from pre‐existing peroxisomes. This apparent paradox has been explained for similar human mutant cell lines (from patients with Zellweger syndrome) by the discovery of peroxisomal membrane ghosts in the mutant cells (Santos, M. J., T. Imanaka, H. Shio, G. M. Small and P. B. Lazarow. 1988. Science 239, 1536–1538). Introduction of a wild‐type gene is thought to restore to the ghosts the ability to import matrix proteins, and thus lead to the refilling of the peroxisomes. It is vitally important to our understanding of peroxisome biogenesis to determine whether the yeast mutants contain ghosts. We have solved this problem by introducing an epitope‐tagged version of Pas3p, a peroxisome integral membrane protein (that is essential for peroxisome biogenesis). Nucleotides encoding a nine amino acid HA epitope were added to the PAS3 gene immediately before the stop codon. The tagged gene (PAS3HA) was inserted in the genome, replacing the wild‐type gene at its normal locus. It was fully functional (the cells assembled peroxisomes normally and grew on oleic acid) but the expression level was too low to detect the protein with monoclonal antibody 12CA5. PAS3HA was expressed in greater quantity from an episomal plasmid with the CUP1 promoter. The gene product, Pas3pHA, was detected by immunogold labelling on the membranes of individual and clustered peroxisomes; the clusters appeared as large spots in immunofluorescence. PAS3HA was similarly expressed in peroxisome biogenesis mutants peb2 and peb4, which lack morphologically recognizable peroxisomes. Gold‐labelled membranes were clearly visible in both mutants: in peb2 the labelled membrane vesicles were generally much smaller than those in peb4, which resembled normal peroxisomes in size.
VIV. Yeast sequencing reports. Sequencing analysis of a 24·7 kb fragment of yeast chromosome XIV identifies six known genes, a new member of the hexose transporter family and ten new open reading frames
Authors: Maftahi M. , Nicaud J.‐M. , Levesque H. , Gaillardin C. 
Published: May 2005

AbstractThe DNA sequence of a 24·7 kb region covering the left arm of chromosome XIV from Saccharomyces cerevisiae was determined. This region contains 17 open reading frames (ORFs) which code for proteins of more than 100 amino acids. Five ORFs correspond to the KRE1, ATP11, DAL82, RFA2 and MCK1 loci, described previously. Two ORFs present high similarity to known proteins: NO345 with the hexose transporter family, and NO351 with the yeast chorismate mutase/prephenate dehydratase enzyme encoded by PHA2. Six ORFs show limited similarity with known proteins or some specific features: NO339 presents 11 potential transmembrane domains. NO343, which is internal to NO345, presents a putative signal sequence and a potential transmembrane domain. NO348 shows similarity with YCW2, TUP1 and SEC13. NO364 reveals a signature for a pyridoxal‐phosphate attachment site. Finally, NO384 and NO388 present a biased amino acid composition, being rich in Asn or Glu/Lys/Arg, respectively. Four other ORFs (NO342, NO376, NO381 and NO397) show no similarity to proteins within the databases screened. The sequence has been entered in the EMBL data library under Accession Number Z46259.
UVS112—A gene involved in excision repair of yeast
Authors: Kozhina Tatiana , Kozhin Sergey , Stepanova Vera , Yarovoy Boris , Donich Vladimir , Fedorova Irina , Korolev Vladimir 
Published: May 2005

AbstractIn this study we show that the previously described uvs112 (uvs12) mutation blocks one of the steps of the excision repair pathway. The properties of this mutation permit the assignment of the UVS112 gene to the RAD3 epistasis group. It was established that the uvs112 mutation caused a 2·5‐fold reduction in the number of recombinants produced by conversion and also significantly increased the frequency of mitotic crossing‐over in interplasmid recombination. Tetrad analysis placed the UVS112 gene on the left arm of chromosome IX, approximately 20 cM from HIS5. The analysis of mitotic recombination revealed that UVS112 lies between HIS6 and HIS5, and is an allele of the RAD25 gene.
Restoration of peroxisome formation in two conditional peroxisome‐deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources
Authors: Titorenko Vladimir I. , Evers Melchior E. , Van Der Klei Ida J. , Harder Wim , Veenhuis Marten 
Published: May 2005

AbstractExpression of the peroxisome‐deficient (Per−) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature‐sensitive (ts) mutants, per13‐6ts and per14‐11ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild‐type cells, namely D‐alanine (for both mutants) or methylamine (for per14‐11ts). These organelles displayed normal wild‐type properties with respect to morphology, mode of development and protein composition.However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D‐alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.