bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of the SPT23 gene: A dosage‐dependent suppressor of ty‐induced promoter mutations from Saccharomyces cerevisiae
Authors: Burkett Thomas J. , Garfinkel David J.
Published: May 2005

SPT genes are suppressors of mutations induced by the retrotransposon Ty in Saccharomyces cerevisiae. All SPT genes isolated to date suppress Ty‐induced mutations by altering transcription. SPT23 was identified as a multicopy suppressor of the Ty‐induced promoter mutations his4‐912δ and lys2‐61. Multicopy expression of SPT23 suppresses a variety of Ty‐induced promoter mutations, including the MAT‐regulated alleles his4‐917 (480) and lys2‐173R2. Here, we report the initial characterization of the SPT23 gene, including its nucleotide sequence and location in the yeast genome. The SPT23 gene contains a 1854 base pair open reading frame. Searches of the current data bases show no homology between SPT23 and previously described genes or proteins. The SPT23 gene is located between RAM2 and MAK11 on the left arm of chromosome XI. Tn10‐LUK insertional mutagenesis of the SPT23 gene indicates that SPT23 is not essential for vegetative growth and spt23 mutations do not confer an Spt− phenotype.
Erratum to: Sequence of a 4.8 kb fragment of Saccharomyces cerevisiae chromosome II including three essential open reading frames
Authors: Baur Axel , Schaaff‐Gerstenschläger Ine , Boles Eckhard , Miosga Thomas , Rose Matthias , Zimmermann Friedrich K.
Published: May 2005

The above paper (Yeast 9:, 289–293, 1993) erroneously presented a non‐updated sequence due to data‐transmission errors. The corrected sequence is 4939 bp in length and has been deposited in the EMBL Data Library under the accession number X71329. Consequently the amino acid sequences of YBR 12.03 and YBR 12.05 changed.
VI. Yeast sequencing reports. The sequence and potential regulatory elements of the HEM2 promoter of Saccharomyces cerevisiae
Authors: Schlaepfer Isabel R. , Mattoon James R. , Bajszár George
Published: May 2005

This paper reports the 1890‐bp sequence located upstream of the HEM2 gene of Saccharomyces cerevisiae. The following potential regulatory protein‐binding motifs were found: ABF1‐binding site, yAP1‐binding site, two REB1‐binding sites, a cyclic AMP‐responsive element, RAP1‐binding site, and several HAP2‐HAP3‐HAP4 binding sites, implicating a complex regulatory mechanism governing expression for the HEM2 gene.
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

Using 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

The 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

We 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

We 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

The 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

The 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

THI4, 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

A 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

A 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

We 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

The 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

The 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

The 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

A 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

Production 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

A 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

We 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.
Sequence of a 17·1 kb DNA fragment from chromosome X of Saccharomyces cerevisiae includes the mitochondrial ribosomal protein L8
Authors: Vandenbol Micheline , Durand Patrick , Dion Caroline , Portetelle Daniel , Hilger Francois
Published: May 2005

We have sequenced a continuous segment of 17 137 bp on chromosome X. Sequence analysis of this stretch revealed 14 open reading frames (ORFs) at least 100 amino acids long. One gene, encoding the mitochondrial 60S ribosomal protein L8, had already been sequenced. Four ORF products show weak homologies with known protein sequences. The nine remaining ORF products have no homologies with sequences in data banks. The nucleotide sequence of the 17·1 kb fragment is available through the EMBL data library under Accession Number Z34288.
Nucleotide sequence and analysis of the centromeric region of yeast chromosome IX
Authors: Voss H. , Tamames J. , Teodoru C. , Valencia A. , Sensen C. , Wiemann S. , Schwager C. , Zimmermann J. , Sander C. , Ansorge W.
Published: May 2005

We have determined the nucleotide sequence of a cosmid (pIX338) containing the centromere region of yeast (Saccharomyces cerevisiae) chromosome IX. The complete nucleotide sequence of 33·8 kb was obtained by using an efficient directed sequencing strategy in combination with automated DNA sequencing on the A.L.F. DNA sequencer. Sequence analysis revealed the presence of 17 open reading frames (ORFs), four of them previously known yeast genes (sly12, pan1, sts1 and prl1), a tRNA gene and the centromere motif. Exhaustive database searches detected sequence homologues of known function for as many as 14 of the 17 ORFs. These include a mammalian tyrosine kinase substrate; the Escherichia coli cell cycle protein MinD; the human inositol polyphosphate‐5‐phosphatase (gene OCRL) involved in Lowe's syndrome, a developmental disorder; and helicases, for which the new yeast member defines a distinct DEAD/H‐box subfamily. A surprisingly large fraction of the ORFs (at least six out of 17) in the centromeric region are apparently involved in RNA or DNA binding.

The nucleotide sequence reported here has been submitted to the EMBL data library under the accession number X79743.
Development and application of an in vivo system to study yeast ribosomal RNA biogenesis and function
Authors: Venema Jaap , Dirks‐Mulder Anita , Faber Alex W. , Raué Hendrik A.
Published: May 2005

We have developed a system for mutational analysis of Saccharomyces cerevisiae ribosomal RNA in vivo in which yeast cells can be made completely dependent on mutant rRNA and ribosomes by a simple switch in carbon source. The system is based on a yeast strain defective in RNA polymerase I (Pol I) transcription [Nogi et al. (1991). Proc. Natl. Acad. Sci. USA 88, 3962–3966]. This normally inviable strain was rescued by integration of multiple copies of the complete 37S pre‐rRNA operon under control of the inducible, Pol II‐transcribed GAL7 promoter into the rDNA repeat on chromosome XII. The resulting YJV100 strain can only grow on medium containing galactose as the carbon source. A second, episomal vector was constructed in which the rDNA unit was placed under control of the constitutive PGK1 promoter. YJV100 cells transformed with this vector are now also able to grow on glucose‐based medium making the cells completely dependent on plasmid‐encoded rRNA. We show that the Pol II‐transcribed pre‐rRNA is processed and assembled similarly to authentic Pol I‐synthesised pre‐rRNA, making this ‘in vivo Pol II system’ suitable for the detailed analysis of rRNA mutations, even highly deleterious ones, affecting ribosome biogenesis or function. A clear demonstration of this is our finding that an insertion into variable region V8 in 17S rRNA, previously judged to be neutral with respect to processing of 17S rRNA, its assembly into 40S subunits and the polysomal distribution of these subunits [Musters et al. (1989), Mol. Cell. Biol. 9, 551–559], is in fact a lethal mutation.
Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated, sorted and matured in the fission yeast Schizosaccharomyces pombe
Authors: Simeon Angela , Egner Ralf , Gascon Santiago , Suarez‐Rendueles Paz
Published: May 2005

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPYsc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPYsc produced in the fission yeast has a higher molecular mass than mature CPYsc produced by the budding yeast. CPYsc is glycosylated when expressed in S. pombe and uses four N‐linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPYsc produced by S. cerevisiae. CPYsc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPYsc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.
Induced expression of bacterial β‐glucosidase activity in saccharomyces
Authors: Adam Ana C. , Rubio‐Texeira Marta , Polaina Julio
Published: May 2005

The bglA gene which encodes a β‐glucosidase from Bacillus polymyxa, has been expressed in Saccharomyces cerevisiae under control of the yeast CYC‐GAL promoter. Strains have been constructed which carry the gene in different locations: in a multicopy plasmid, a single integration at the URA3 locus, or multiple integrations at the RDN1 locus. Integrative transformation at RDN1 yielded genetically stable clones with a high level of β‐glucosidase activity. Coordinated overexpression of the GAL4 inducer protein further increased the level of enzyme activity, although eventually caused the lysis of the cultures. Diploid, triploid and tetraploid strains derived from the transformants with multiple integrations were constructed and expression of β‐glucosidase activity in different conditions of growth was assayed. While per‐cell activity increased with ploidy, specific activity was about the same in strains of equivalent genotype regardless of ploidy. Genetically stable and regulated expression in Saccharomyces of β‐glucosidase activity is interesting for the development of strains able to ferment β‐glycosidic sugars (i.e. cellobiose and lactose). From another point of view, the bglA product proved to be a convenient reporter enzyme to monitor heterologous gene expression.
Saccharomyces cerevisiae mRNA 3′ end forming signals are also involved in transcription termination
Authors: Russo Patrick
Published: May 2005

Previously, a 38‐base‐pair (bp) region in the 3′ untranslated portion of the Saccharomyces cerevisiae iso‐1‐cytochrome c gene, was shown to be required for both normal CYC1 mRNA 3′ end formation (Zaret and Sherman, 1982), and efficient transcription termination (Russo and Sherman, 1989). In another study, specific sequences such as TATATA, TACATA, and TAGTAGTA were shown to be involved in mRNA 3′ end formation in S. cerevisiae (Russo et al., 1991). In this report, an in vivo plasmid stability assay has been utilized to show that these and related sequences are also involved in transcription termination, at varying efficiencies, and in an orientation‐dependent manner. For example: the sequence TATATA appeared to terminate transcription almost as efficiently as the original wild type 38‐bp region; whereas, the sequences TAGATATATGTAA and TACATA were less efficient, and TTTTTTTATA had little, if any, transcription termination function. In contrast, none of these sequences appeared to terminate transcription in the reverse orientation. Therefore, it appears that certain sequence signals capable of promoting mRNA 3′ end formation in yeast, are also directly involved in transcription termination.
Sequence and analysis of 24 kb on chromosome II of Saccharomyces cerevisiae
Authors: Aljinovic Gordana , Pohl Thomas M.
Published: May 2005

In the course of the European yeast genome sequencing project, we determined 23,920 bp of a continuous chromosome II right arm sequence. Analysis of data revealed 13 open reading frames (ORFs), three of which corresponded to previously identified genes; two tRNA genes and one repetitive element. One ORF showed considerable homology (46%) to a hypothetical chromosome III gene; another, putatively very hydrophobic gene product, was 30% identical to the heat‐shock protein HSP30. Two ORFs were homologous to human genes. The complete sequence was submitted to the EMBL data bank under the Accession Number Z46260 Authorin submission ‘3’.
Mutations sensitizing yeast cells to the start inhibitor nalidixic acid
Authors: Prendergast John A. , Singer Richard A. , Rowley Neil , Rowley Adele , Johnston Gerald C. , Danos Maria , Kennedy Brian , Gaber Richard F.
Published: May 2005

The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal‐sensitive mutations were sought because the inhibitory effects of Nal on wild‐type cells are only transient, and wild‐type cells naturally become refractory to Nal. Three complementation groups of Nal‐sensitive mutations were found. Mutations in the first complementation group were shown to reside in the ARO7 gene, encoding chorismate mutase; tyrosine and phenylalanine synthesis was inhibited by Nal in these aro7 mutants, whereas wild‐type chorismate mutase was unaffected. These aro7 alleles demonstrate ‘recruitment’, by mutation, of an innately indifferent protein to an inhibitor‐sensitive form. The Nal‐sensitive aro7 mutant cells were used to show that the resumption of Nal‐inhibited nuclear activity and cell proliferation takes place while cytoplasmic Nal persists at concentrations inhibitory for the mutant chorismate mutase. Mutations in the second complementation group, nss2 (Nal‐supersensitive), increased intracellular Nal concentrations, and may simply alter the permeability of cells to Nal. The third complementation group was found to be the ERG6 gene, previously suggested to encode the ergosterol biosynthetic enzyme sterol methyltransferase. Mutation or deletion of the ERG6 gene had little effect on the inhibition of Start by Nal, but prevented recovery from this inhibition. Mutation of ERG3, encoding another ergosterol biosynthetic enzyme, also caused Nal sensitivity, suggesting that plasma membrane sterol composition, and plasma membrane function, mediates recovery from Nal‐mediated inhibition of Start.
The peculiarities of the interaction of radiation and hyperthermia in Saccharomyces cerevisiae irradiated with various dose rates
Authors: Petin Vladislav G. , Zhurakovskaya Galina P.
Published: May 2005

In experiments with yeast cells it was shown that the synergistic effect of a simultaneous action of ionizing radiation and hyperthermia was a function of dose rate. It was demonstrated that, with increasing dose rate to obtain maximum synergistic effect, the temperature at which radiation is delivered should be elevated. Possible explanation of this effect is discussed.
Automatic intron detection in nuclear DNA sequences of Saccharomyces cerevisiae
Authors: Kalogeropoulos Angelos
Published: May 2005

The goal of the present work is the construction of software (EXPLORA) which automatically detects Open Reading Frames (ORF), intron‐containing or not, in the nuclear sequences of the yeast Saccharomyces cerevisiae. In order to detect introns within (or preceding) ORFs, one must define precise rules of how an intron is identified. These rules are described here. The software is able to detect correctly an intron‐associated ORF in at least 88% of cases. We tested all yeast nuclear entries of the EMBL database with the software, and found, in addition to the known intron‐associated ORFs, some others which may include an intron. Finally the software was applied to the DNA sequences of chromosomes III and XI, in which it detected eight new intron‐associated ORFs.
Identification and initial characterization of the cytosolic protein Ycr77p
Authors: Rodriguez‐Cousino Nieves , Lill Roland , Neupert Walter , Court Deborah A.
Published: May 2005

The nucleotide sequence of yeast chromosome III encompassing the previously described open reading frames (ORFs) YCR80w, YCR77c and YCR78c (Oliver et al., 1992) has been updated. In the corrected sequence, these ORFs are replaced by two new ORFs, YCR80w (453 bp) and YCR77c (2391 bp). In addition, the orientation of Ycr79c is reversed to give ORF Ycr79w, which has an unaltered nt sequence. The predicted translation products do not exhibit significant homology to known proteins. ORF Ycr77p encodes an 88 kDa, cytosolic protein. A fraction of the protein is associated with small membranous structures in a salt‐sensitive fashion. Initial characterization revealed that the protein is not essential for yeast viability, growth on non‐fermentable carbon sources, mating and sporulation. The chromosome III DNA sequence that was used for the analysis has the Accession Number X59720 in the GenBank/EMBL database.
The maintenance of self‐replicating plasmids in Saccharomyces cerevisiae: Mathematical modelling, computer simulations and experimental tests
Authors: Van Der Sand Sueli T. , Greenhalf William , Gardner David C. J. , Oliver Stephen G.
Published: May 2005

A distributive model has been constructed to describe the maintenance of the native 2 μm and 2 μm‐based plasmids in the yeast Saccharomyces cerevisiae. This model includes elements which represent the influence of selection, segregation, replication and amplification on plasmid stability. A computer program has been written in TURBO PASCAL to implement the model and a number of simulation experiments have been carried out. These simulations permitted the choice of a form of the model which is compatible with the available experimental evidence. The form chosen involves an amplification system in which the RAF gene product binds to the Rep1/Rep2 dimer to prevent the latter acting to repress the activity of the FLP gene. At the same time an upper limit (or ‘ceiling’) was imposed on the number of plasmid molecules able to replicate. Maternal bias was accommodated by ‘tagging’ a small proportion of molecules for inheritance by the mother nucleus and these tags being removed (or ‘cleared’) by the Rep1/Rep2 dimers. This final form of the model makes specific predictions about the stability of 2 μm and YEp plasmids in yeast populations and about the distribution of plasmid copy number between cells in such populations. The predictions on stability have been subjected to experimental test and results provide good support for the model.
High‐Resolution cosmid mapping of the left arm of Saccharomyces cerevisiae chromosome XII; A first step towards an ordered sequencing approach
Authors: Scholler Patrik , Schwarz Sandra , Hoheisel Jörg D.
Published: May 2005

For the sequencing of the left arm of chromosome XII of Saccharomyces cerevisiae, we fine‐mapped the entire 450 kb fragment between the ribosomal DNA (rDNA) and the left telomere. Total yeast DNA in agarose blocks was digested with I‐PpoI, which exclusively cuts once in each repeat unit of the rDNA. The resulting fragment was isolated from pulsed‐field gels, together with the equally sized chromosome IX. A cosmid library of some 30‐fold chromosome coverage was generated from this material, with the cloning efficiency being around 20 000 clones per microgram genomic DNA. The chromosome XII and IX specific clones were identified by complementary hybridizations with the respective chromosomes. For the left arm of chromosome XII, a contiguous cosmid array (contig) with an average map resolution better than 9 kb was generated by clone hybridization procedures. The ordered library serves as a tool for the physical mapping of genetic markers. Also, a minimal set of 15 clones was selected that covers the entire fragment. This subset forms the basis for the generation of a template map of much higher resolution for a directed sequencing of the left arm of chromosome XII.
Mapping of the ACC1/FAS3 gene to the right arm of chromosome XIV of Saccharomyces cerevisiae
Authors: Guerra Cesar E. , Klein Hannah L.
Published: May 2005

The ACC1/FAS3 gene has been mapped to the right arm of chromosome XIV by both genetic and physical methods. The gene is closely linked to RNA2 and is allelic to the ABP2 gene of chromosome XIV.
Characterization of the glyceraldehyde‐3‐phosphate dehydrogenase gene family from Kluyveromyces marxianus—polymerase chain reaction–single‐strand conformation polymorphism as a tool for the study of multigenic families
Authors: Fernandes P. A. , Sena‐Esteves M. , Moradas‐Ferreira P.
Published: May 2005

Three glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) genes from Kluyveromyces marxianus were identified and characterized. The coding region of two of them (GAP2 and GAP3) is very similar (99·6% homology). The other gene (GAP1) is only 86% homologous to GAP2 or GAP3 and is responsible for the expression of Gap1p. This protein is extremely homologous to the K. marxianus cell wall protein p37, presumably involved in flocculation. However, no leader sequence could be detected in this gene. The identification of the three genes was possible with the use of polymerase chain reaction–single‐strand conformation polymorphism (PCRSSCP), as it permits us to overcome the difficulties caused by the high homology amongst the genes. Expression of the GAPDH genes under different carbon sources (glucose or ethanol) was assessed either by Northern blot or reverse transcription–PCRSSCP analysis, revealing that genes GAP1 and GAP2, but not GAP3, are transcribed. The results also indicate that the transcription of the gene encoding the cell wall protein p37 (Gap1p) is not dependent on the carbon source, in contrast with the expression of the gene GAP2, which is affected in cells growing in a glucose‐depleted medium.
The S1, S2 and SGA1 ancestral genes for the STA glucoamylase genes all map to chromosome IX in Saccharomyces cerevisiae
Authors: Lambrechts Marius G. , Pretorius Isak S. , Marmur Julius , Sollitti Paul
Published: May 2005

The polymorphic extracellular glucoamylase‐encoding genes STA1 (chr. IV), STA2 (chr. II) and STA3 (chr. XIV), from Saccharomyces cerevisiae var. diastaticus probably evolved by genomic rearrangement of DNA regions (S1, S2 and SGA1) present in S. cerevisiae, and subsequent translocation to unlinked regions of chromosomal regions. S1, encoding a homologue to the threonine/serine‐rich domain of STA glucoamylases (GAI‐III), mapped to the right arm of chromosome IX. S2, encoding the hydrophobic leader peptide of GAI‐III, was also mapped on the right arm of chromosome IX, next to S1, close to DAL81. The SGA1 sporulation‐specific, intracellular glucoamylase‐encoding gene is located on the left arm of chromosome IX, 32 kb proximal of HIS5.
A 37·5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes, a TCP‐1‐related gene, an open reading frame similar to the DAL80 gene, and a tRNAArg
Authors: Rasmussen Søren W.
Published: May 2005

The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromsome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor‐like protein, D678 is identical to the yeast GSH1 gene encoding γ‐glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Ccη subunit of a murine TCP‐1‐containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP‐1 family. Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNAArg3 (AGC) gene. The nucleotide sequence data reported in this paper have been deposited in the EMBL and GenBank databases under the accession number X85021.
II. Yeast sequencing reports. Sequence analysis of a 78·6 kb segment of the left end of Saccharomyces cerevisiae chromosome II
Authors: Obermaier Brigitte , Gassenhuber Johann , Piravandi Ester , Domdey Horst
Published: May 2005

We report the sequence analysis of a 78,601 bp DNA segment on the left arm of chromosome II of Saccharomyces cerevisiae. This 78·6 kb segment spans the region from the start of a subtelomeric Y′ element up to the ILS1 gene. It contains 49 open reading frames (ORFs) with more than 100 amino acids length including 14 internal and five overlapping ORFs. The gene density, excluding the internal ORFs, was calculated as one ORF per 2·2 kb. Eight ORFs (PKC1, TyA, TyB, ATP1, ROX3, RPL17a, PET112 and ILS1) correspond to previously characterized genes. ORF YBL0718 was identified as CDC27; YBL0706 as TEL1. Four other ORFs show strong similarities to already known genes. The gene product of YBL0838 is 60% identical to the ribosomal protein RPL32 from rat, mouse and man. YBL0701 encodes a protein with significant similarity to the initiation factor eIF2 associated p67 glycoprotein from rat. Eight ORFs were disrupted and the resulting yeast strains analysed with respect to their phenotype. The sequence has been deposited in the EMBL Nucleotide Sequence Database under the Accession Number X79489.