bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physiological characterization of the yeast metallothionein (CUP1) promoter, and consequences of overexpressing its transcriptional activator, ACE1
Authors: Hottiger Thomas , Fürst Peter , Pohlig Gabriele , Heim Jutta 
Published: May 2005

AbstractUsing the anticoagulant, hirudin, from the leech Hirudo medicinalis as a secreted reporter protein, the influence of physiological parameters on activity and regulation of the yeast (Saccharomyces cerevisiae) metallothionein (CUP1) promoter was studied. Induction of CUP1‐directed hirudin expression from 2μ‐based vectors was possible at any time point during diauxic batch growth, even in cells approaching stationary phase. The highest titers of hirudin were obtained when the CUP1 promoter was activated immediately following inoculation of the cultures. If such a pseudo‐constitutive fermentation strategy was adopted, the promoter was superior to an optimized variant (GAPFL) of the strong, constitutive GAPDH promoter. This superiority was primarily due to the relative independence of CUP1 promoter activity of the physiological status of host cells: whilst the maximal strength of the CUP1 and GAPFL promoters was comparable, CUP1‐directed hirudin expression was high in all phases of diauxic batch growth, whereas hirudin production from the GAPFL promoter declined in post‐diauxic cultures. High activity of the CUP1 promoter was observed on both a fermentable (glucose) and a non‐fermentable (ethanol) carbon source. Hirudin expression could be adjusted to different levels by varying the amount of inducer (cupric sulphate) added to cultures. The copper concentrations required for maximal promoter induction had no negative effects on host growth and interfered with neither hirudin secretion nor with the biological activity of the peptide. Overexpression of the transcriptional activator, ACE1, resulted in increased levels of hirudin mRNA. Hirudin titers increased in parallel to mRNA concentrations in cultures grown in the presence of low concentrations of copper. In contrast, at high copper doses, elevated levels of the ACE1 protein resulted in inferior hirudin production. Cells overexpressing ACE1 while harbouring a CUP1‐drived hirudin expression cassette showed slow growth and poor plasmid maintenance. It was tested whether this might be the result of a block in the secretory pathway; however, measurements of intracellular hirudin did not support this hypothesis. The data rather indicated that hirudin production was limited by a metabolic constraint downstream of transcription but upstream of the secretory pathway.
Promoter analysis of the PDA1 gene encoding the E1α subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae
Authors: Wenzel Thibaut J. , Zuurmond Anne‐Marie , Bergmans Anneke , Van Den Berg Johan A. , Yde Steensma H. 
Published: May 2005

AbstractThe location and sequence of the PDA1 gene, encoding the E1α subunit of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cerevisiae, were determined. The PDA1 gene was located on a 6·2 kb fragment of chromosome V, approximately 18 kb centromere distal to RAD3. Consistent with this, the PDA1 gene was genetically mapped at 4 cM from RAD3. A part of the 6·2 kb fragment of chromosome V was sequenced. The nucleotide sequence contained the PDA1 open reading frame and the entire putative promoter. Computer analysis revealed a putative GCN4 binding motif in the PDA1 promoter. The presence of transcriptional elements was experimentally determined by deletion analysis. To this end, ExoIII deletions were constructed in the 5′ to 3′ direction of the PDA1 promoter and effects on transcription were determined by Northern analysis. Transcription was unaffected upon deletion to position − 190 relative to the ATG start codon. Deletions from position − 148 and beyond, however, reduced promoter activity at least 40‐fold. Apparently the 42 bp between nucleotides − 190 and − 148 contain an element essential for transcription. Inactivation of the PDA1 promoter could not be attributed to deletions of a recognizable TATA element or any known yeast regulatory motifs. The possible role of the CCCTT sequence present in the 42 bp region and also in the promoters of the other genes encoding subunits of the PDH complex is discussed.
A new family of yeast genes implicated in ergosterol synthesis is related to the human oxysterol binding protein
Authors: Jiang Bo , Brown Jeffrey L. , Sheraton Jane , Fortin Nathalie , Bussey Howard 
Published: May 2005

AbstractWe have identified three yeast genes, KES1, HES1 and OSH1, whose products show homology to the human oxysterol binding protein (OSBP). Mutations in these genes resulted in pleiotropic sterol‐related phenotypes. These include tryptophan‐transport defects and nystatin resistance, shown by double and triple mutants. In addition, mutant combinations showed small but apparently cumulative reductions in membrane ergosterol levels. The three yeast genes are also functionally related as overexpression of HES1 or KES1 alleviated the tryptophan‐transport defect in kes1Δ or osh1Δ mutants, respectively. Our study implicates this new yeast gene family in ergosterol synthesis and provides comparative evidence of a role for human OSBP in cholesterol synthesis.
Selective retention of secretory proteins in the yeast endoplasmic reticulum by treatment of cells with a reducing agent
Authors: Jämsä Eija , Simonen Marjo , Makarow Marja 
Published: May 2005

AbstractWe have used four glycoproteins as markers to study how disulfide bond formation and protein folding effect the intracellular transport of proteins in yeast. Under normal conditions, the vacuolar enzyme carboxypeptidase Y (CPY) and the secretory stress‐protein hsp150 acquired disulfide bonds in the endoplasmic reticulum (ER). Treatment of living cells with the reducing agent dithiothreitol (DTT) prevented disulfide formation of newly synthesized CPY and hsp150, resulting in retention of the proteins in the ER. When DTT was removed, the sulfhydryls were reoxidized, and the transport of the proteins to their correct destinations was resumed. Even mature CPY, located in the vacuole, could be reduced with DTT, and reoxidized after removal of the drug. DTT treatment blocked intracellular transport of hsp150 only when present during the synthesis and translocation of the protein. Reduction of folded hsp150, accumulated in the ER due to a sec block prior to DTT treatment, did not inhibit its secretion. The Kar2p/BiP protein, a component of the ER lumen, was found to be associated with fully translocated reduced hsp150, but not with native hsp150, suggesting that Kar2p/BiP may be involved in the putative retention mechanism. The cysteine‐free pro‐α‐factor, and invertase which was shown to have free sulfhydryls, were secreted and modified similarly in the presence and absence of DTT, showing that the secretory pathway of yeast functioned under reducing conditions.
Yeast sequencing reports. Isolation and sequence analysis of a gene from the linear DNA plasmid pPAC1–2 of Pichia acaciae that shows similarity to a killer toxin gene of Kluyveromyces lactis
Authors: Bolen P. L. , Eastman E. M. , Cihak P. L. , Hayman G. T. 
Published: May 2005

AbstractThe toxin‐encoding linear plasmid systems found in Pichia acaciae and Kluyveromyces lactis yeasts appear to be quite similar, both in function and structural organization. By Southern hybridization, a linear plasmid of P. acaciae, pPac1–2, was found to hybridize to the second open reading frame (ORF2) of K. lactis plasmid pGKL1, known to encode the α and β subunits of the K. lactis toxin. A 1·7 kbp segment of pPac1–2 DNA was cloned, sequenced and shown to contain four regions of strong homology to four similarly oriented regions of K. lactis ORF2. This 1·7 kbp fragment also contained an ORF of 1473 bp that could encode a protein of ∼ 55·8 kDa. Like the α subunit gene of K. lactis ORF2, a very hydrophobic region occurs at the N‐terminus, perhaps representing a signal sequence for transport out of the cell. Unlike K. lactis ORF2, however, the encoded polypeptide is much smaller and lacks a recognizable domain common to chitinases. The structure of a toxin that includes the translation product of this P. acaciae ORF would likely be quite different from that of the K. lactis toxin. Analysis of the upstream region of the P. acaciae ORF revealed an upstream conserved sequence identical to that found before ORFs 8 and 9 of pGKL2. A possible hairpin loop structure, as has been described for each of the four K. lactis pGKL1 ORFs, was found just upstream of the presumed start codon. The similarity of the promoter‐like elements found in the linear plasmid genes of these diverse yeasts reinforces the idea of the existence of a unique, but highly conserved, expression system for these novel plasmids. The sequence has been deposited in the GenBank data library under Accession Number U02596.
Nadh: Ubiquinone oxidoreductase in obligate aerobic yeasts
Authors: Büschges Rainer , Bahrenberg Gregor , Zimmermann Martin , Wolf Klaus 
Published: May 2005

AbstractThe strictly aerobic yeasts Candida pinus, Cryptococcus albidus, Rhodotorula minuta, Rhodotorula mucilaginosa and Trichosporon beigelii possess mitochondrial NADH dehydrogenases with significant features of the NADH:ubiquinone oxidoreductase (complex I). These species show in all growth phases and under standard cultivation conditions, NADH dehydrogenases of approximately 700 kDa, which are sensitive to rotenone, a specific inhibitor of this complex. Identical results were obtained with the weakly fermenting C. pinus. The facultatively fermenting yeasts Saccharomyces cerevisiae and Kluyveromyces marxianus do not possess the 700 kDa‐complex and are insensitive to rotenone. In S. cerevisiae, a rotenone‐insensitive NADH dehydrogenase of about 500–600 kDa is detected only in stationary phase cells.As in Neurospora crassa, upon incubation of the obligately aerobic yeast R. mucilaginosa with chloramphenicol, an intermediate NADH dehydrogenase of approximately 350 kDa was formed, which was insensitive to rotenone.
Regulation of THI4(MOL1), a thiamine‐biosynthetic gene of Saccharomyces cerevisiae
Authors: Praekelt Uta M. , Byrne Kerry L. , Meacock Peter A. 
Published: May 2005

AbstractTHI4, a Saccharomyces cerevisiae gene originally identified as a result of transient expression in molasses medium and named MOL1 is regulated by thiamine. Using a THI4 promoter‐lacZ fusion on a centromeric yeast vector, we have shown that the THI4 is completely repressed throughout batch culture by thiamine at a concentration around 1 μM, but shows high level constitutive expression in thiamine‐free medium. The transient expression pattern observed in molasses medium can be mimicked by the addition of 0·15 μM‐thiamine to defined minimal medium. Cells grown in thiamine‐free medium have an intracellular thiamine concentration of around 9 pmol/107 cells. A low level (1 μM) of exogenous thiamine is completely sequestered from the medium within 30 min; intracellular thiamine concentrations rise rapidly, followed by a gradual decrease as a result of dilution during growth. A saturating extracellular level of thiamine leads to a maximal intracellular concentration of around 1600 pmol/107 cells, at which point the transport system is shut down. After transfer from repressing to non‐repressing medium, THI4 becomes induced when the intracellular concentration of thiamine falls to 20 pmol/107 cells. A thi4::UARA3 disruption strain is auxotrophic for thiamine, but can grow in the presence of hydroxyethyl thiazole, indicating that the gene product is involved in the biosynthetic pathway leading to the formation of the thiazole precursor of thiamine.
The Saccharomyces cerevisiae gene PPH3 encodes a protein phosphatase with properties different from ppx, PP1 and PP2A
Authors: Hoffmann Ralf , Jung Stephan , Ehrmann Michael , Hofer Hans Werner 
Published: May 2005

AbstractA clone encoding the catalytic subunit of a protein phosphatase from Saccharomyces cerevisiae was isolated. Except for replacement of IIe‐245 by Met the structure of the phosphatase was identical to that encoded by PPH3 (Ronne, H., Carlberg, M., Hu, G. Z. and Nehlin, J. O. (1991). Mol. Cell. Biochem. 11, 4876–4884) and exhibited 63% sequence identity to PPX cloned from a rabbit liver cDNA library (Brewis, N. D., Street, A. J., Prescott, A. R. and Cohen, P. T. W. (1993). EMBO J. 12, 987–996). Expression of active enzyme was achieved in Escherichia coli mutants which were generated by a genetic selection based on functional complementation of bacterial phosphoserine phosphatase. Though some of the properties of PPH3 resembled those of protein phosphatase 2A and PPX, others were different. PPH3 exhibited lower sensitivity against inhibition by okadaic acid, showed different substrate specificity and required a divalent cation (Mn2+ was preferred before Mg2+ and Ca2+) for activity when assayed with phospho‐histone as a substrate. However, 25% of maximum activity was observed in the absence of divalent cations when the peptide LRRAS(P)LG was used as substrate. The PPH3‐protein was also identified by chromatography of extracts from S. cerevisiae on DEAE–cellulose. Protein immunoreactive with an antiserum raised against the non‐conserved N‐terminal 53 amino acids of PPH3 was coeluted with a single peak of LRRAS(P)LG dephosphorylating activity.
Yeast sequencing reports. APL1, a yeast gene encoding a putative permease for basic amino acids
Authors: Sychrova Hana , Chevallier Marie‐Renee 
Published: May 2005

AbstractA Saccharomyces cerevisiae gene (1722 bp), encoding a protein (574 aa) highly homologous to the basic‐amino‐acid permeases LYP1 and CAN1, was sequenced. The gene, which was named APL1 (Amino‐acid Permase Like), is located 881 bp upstream from LYP1 (lysine‐specific permease), and in head‐to‐head orientation to it. These sequence data have been deposited in the EMBL/GenBank/DDBJ nucleotide sequence data libraries under Accession Number X74069.
Defining the sequence specificity of the Saccharomyces cerevisiae DNA binding protein REB1p by selecting binding sites from random‐sequence oligonucleotides
Authors: Liaw Patricia C. Y. , Brandl Christopher J. 
Published: May 2005

AbstractWe have used a random selection protocol to define the consensus and range of binding sites for the Saccharomyces cerevisiae REB1 protein. Thirty‐five elements were sequenced which bound specifically to a GST‐REB1p fusion protein coupled to glutathione–Sepharose under conditions in which more than 99·9% of the random sequences were not retained. Twenty‐two of the elements contained the core sequence CGGGTRR, with all but one of the remaining elements containing only one deviation from the core. Of the core sequence, the only residues that were absolutely conserved were the three consecutive G residues. Statistical analysis of a nucleotide‐use matrix suggested that the REB1p binding site also extends into flanking sequences with the optimal sequence for REB1p binding being GNGCCGGGGTAACNC. There was a positive correlation between the ability of the sites to bind in vitro and activate transcription in vivo; however, the presence of non‐conformants suggests that the binding site may contribute more to transcriptional activation than simply allowing protein binding. Interestingly, one of the REB1p binding elements had a DNAse 1 footprint appreciably longer than other elements with similar affinity. Analysis of its sequence indicated the potential for a second REB1p binding site on the opposite strand. This suggests that two closely positioned low‐affinity sites can function together as a highly active site. In addition, database searches with some of the randomly defined REB1p binding sites suggest that related elements are commonly found within ‘TATA‐less’ promoters.
X. Yeast sequencing reports. Revised nucleotide sequence of the cor region of yeast Saccharomyces cerevisiae chromosome X
Authors: Huang Meng‐Er , Manus Vladimir , Chuat Jean‐Claude , Galibert Francis 
Published: May 2005

AbstractThe COR region, a gene cluster located on chromosome X of Saccharomyces cerevisiae and including genes CYC1, UTR1, UTR3, OSM1, tRNAGly and RAD7, was sequenced within the framework of the European Union genome systematic sequencing project. It was compared with previously published sequences to be found in GenBank under the acronym YSCCORA. While some of the discrepancies observed can be readily ascribed to polymorphism, others most probably result from sequencing errors. A revised version of the sequence of the COR cluster is given. The sequence has been deposited in the EMBL Data Library under Accession Number L26347.
Formation of poliomyelitis subviral particles in the yeast Saccharomyces cerevisiae
Authors: Jore J. P. M. , Veldhuisen G. , Kottenhagen M. , Pouwels P. H. , Foriers A. , Rombaut B. , Boeyé A. 
Published: May 2005

AbstractThe sequence of the poliovirus genome encoding 3CD (a protease) was transferred to the yeast Saccharomyces cerevisiae on expression vectors with either a constitutive or an inducible promoter. Transformants could only be obtained with vectors carrying the inducible transcription unit. Extracts of induced cells were able to cleave cell‐free synthesized P1, the precursor of the poliovirus capsid proteins, into VP0, VP3 and VP1.In yeast cells constitutively expressing P1, induction of 3CD expression resulted in only trace amounts of processed products. Processing could be improved considerably by simultaneous induction of both P1 and 3CD expression. Analysis of extracts of such induced cells revealed the presence of particles that resembled authentic subviral particles.
I. Yeast sequencing reports. LTE1 of Saccharomyces cerevisiae is a 1435 codon open reading frame that has sequence similarities to guanine nucleotide releasing factors
Authors: Keng Teresa , Clark Michael W. , Storms Reg K. , Fortin Nathalie , Zhong Wuwei , Francis Ouellette B. F. , Barton Arnold B. , Kaback David B. , Bussey Howard 
Published: May 2005

AbstractThe DNA sequence of the LTE1 gene on the left arm of chromosome I of Saccharomyces cerevisiae has been determined. The LTE1 open reading frame comprises 4305 bp that can be translated into 1435 amino acid residues. The position of this open reading frame corresponds well to that of a 4·7 kb transcript that has been mapped to this position. The derived amino acid sequence has significant similarities to the amino acid sequence of the guanine nucleotide releasing factor isolated from a rat brain library. The carboxy‐terminus of the LTE1 protein also shows similarities to other guanine nucleotide exchange factors of the S. cerevisiae CDC25 family. The sequence has been deposited in the GenBank data library under Accession Number L20125.
II. Yeast sequencing reports. Sequence of a segment of yeast chromosome II shows two novel genes, one almost entirely hydrophobic and the other extremely asparagine‐serine rich
Authors: Cusick Michael E. 
Published: May 2005

AbstractA 3·2 kb EcoRI fragment of yeast Saccharomyces cerevisiae was entirely sequenced. Two new open reading frames were identified. The first is extremely hydrophobic, and would likely be an integral membrane protein. It has significant similarity to only one reported gene, a gene of unknown function from Drosophila melanogaster. The second ORF is asparagine‐rich and very serine‐rich, with a remarkable stretch of nearly 26 consecutive asparagine residues comprised of the same codon. It has no significant similarity to any reported gene. The fragment maps to chromosome II on the left arm between the CDC27 and ILS1 loci. The nucleotide sequence reported in this paper has been deposited in the GenBank database with the Accession Number M89908.
Physiological approach to heterologous human serum albumin production by Kluyveromyces lactis in chemostat culture
Authors: Blondeau Karine , Boze Hélène , Jung G. , Moulin G. , Galzy P. 
Published: May 2005

AbstractProduction of recombinant human serum albumin (rHSA) controlled by the constitutive promoter phosphoglycerate kinase was studied in Kluyveromyces lactis. It was governed by both cell concentration and glycolytic flow. The triggering of the fermentation metabolism by unfavourable culture conditions (pH, pO2, D) caused a decrease in the synthesis of the heterologous protein. The highest productivity (75 mg 1−1 per h) and rHSA concentration (62 mg 1−1) were obtained in chemostat culture with a dilution rate of 0·12 h−1 and with 38 g 1−1 dry weight.
XV. Yeast sequencing reports. Sequence of a 10·27 kb segment on the left arm of chromosome XV from Saccharomyces cerevisiae includes part of the IRA2 gene and a putative new gene
Authors: Zumstein Emmanuelle , Griffin Hugh , Schweizer Michael 
Published: May 2005

AbstractA 10 270 bp fragment from the left arm of chromosome XV of Saccharomyces cerevisiae was sequenced and analysed. The sequence reveals the presence of two open reading frames (ORFs), one of them is the larger part of the previously sequenced gene IRA2 (YOL0951). The other ORF, YOL0950, has a length of 1245 nucleotides and exhibits no significant homology with any known gene, although there is some similarity of its upstream region to the corresponding region of the Schizosaccharomyces pombe cdr1/nim1 gene which is involved in the control of mitotic cell size. The sequence has been deposited in the EMBL data library under Accession Number X75449.
2‐μm vectors containing the Saccharomyces cerevisiae metallothionein gene as a selectable marker: Excellent stability in complex media, and high‐level expression of a recombinant protein from a CUP1‐promoter‐controlled expression cassette in cis
Authors: Hottiger Thomas , Kuhla Jochen , Pohlig Gabriele , Fürst Peter , Spielmann Albert , Garn Morten , Haemmerli Stephan , Heim Jutta 
Published: May 2005

AbstractWe have constructed 2‐μm‐based yeast expression vectors containing a copy of the metallothionein (CUP1) gene of Saccharomyces cerevisiae as a semi‐dominant, selectable marker. When used for the expression of the thrombin inhibitor hirudin, originally derived from the leech Hirudo medicinalis, these vectors displayed the following characteristics. (1) In the presence of copper salts, they were mitotically more stable than similarly designed control vectors lacking the CUP1 gene. In copper‐sensitive host strains, the apparent plasmid stability was 100%, even in complex media and during fed‐batch fermentation for an extended period of time. (2) Use of the CUP1‐stabilized plasmids improved the production of hirudin by both copper‐sensitive and copper‐resistant hosts. The highest hirudin titers were obtained with a ΔCUP1 host. (3) Copper selection resulted in a moderate increase in average plasmid copy numbers (up to two‐fold) as assessed by measuring hirudin expression from a constitutive promoter (GAPFL). This effect was most noticeable if the vector showed an asymmetric segregation pattern (i.e., high rates of plasmid loss in the absence of copper). (4) The CUP1 marker proved particularly useful in combination with a CUP1‐promoter‐controlled expression cassette on the same plasmid. In such a set‐up, the rates of transcription of the heterologous protein and that of the selectable marker are tightly linked. Therefore, an increase in selective pressure directly provokes an increase in product yields. In a copper‐sensitive host strain, this plasmid design allowed for the production of very high amounts of biologically active hirudin. Our results clearly establish the utility of the CUP1 marker in the construction of stable yeast expression vectors.