bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibodies directed against a yeast carboxyl‐terminal peroxisomal targeting signal specifically recognize peroxisomal proteins from various yeasts
Authors: Aitchison John D. , Szilard Rachel K. , Nuttley William M. , Rachubinski Richard A.
Published: May 2005

The carboxyl‐terminal tripeptide Ala‐Lys‐Ile is essential for targeting Canadida tropicalis trifunctional enzyme (hydratase‐dehydrogenase‐epimerase) to peroxisomes of both Candida albicans and Saccharomyces cerevisiae (Aitchison, J. D., Murray, W. W. and Rachubinski, R. A. (1991). J. Biol. Chem. 266, 23197–23203). We investigated the possibility that this tripeptide may act as a general peroxisomal targeting signal (PTS) for other proteins in the yeasts C. tropicalis, C. albicans, Yarrowia lipolytica and S. cerevisiae, and in rat liver. Anti‐AKI antibodies raised against the carboxyl‐terminal 12 amino acids of trifunctional enzyme were used to search for this PTS in proteins of these yeasts and of rat liver. The anti‐AKI antibodies reacted exclusively with multiple peroxisomal proteins from the yeasts C. tropicalis, C. albicans and Y. lipolytica. There was a weak reaction of the antibodies with one peroxisomal protein from S. cerevisiae and no reaction with peroxisomal proteins from rat liver. Antibodies directed against a synthetic peptide containing a carboxyl‐terminal Ser‐Lys‐Leu PTS (Gould, S. J., Krisans, S., Keller, G.‐A. and Subramani, S. (1990). J. Cell Biol. 110, 27–34) reacted with multiple peroxisomal proteins of rat liver and with peroxisomal proteins of yeast distinct from those identified with anti‐AKI antibodies. These results provide evidence that several peroxisomal proteins of different yeasts contain a PTS antigenically similar to that of C. tropicalis trifunctional enzyme and that this signal is absent from peroxisomal proteins from at least one mammalian system, rat liver.
III. Yeast sequencing reports. Nucleotide sequence of 9·2 kb left of CRY1 on yeast chromosome III from strain ab972: Evidence for a ty insertion and functional analysis of open reading frame YCR28
Authors: Carbone M. L. Agostoni , Panzeri L. , Falconi M. Muzi , Carcano C. , Plevani P. , Lucchini G.
Published: May 2005

We report the 9210 bp sequence from a segment of yeat chromosome III cloned from strain AB972 in λPM3270. Analysis of this sequence and its comparison with the one derived from the corresponding segment of strain XJ24‐24A revealed that the AB972 region contains a duplication of about 2 kb and a Ty element, which are not found in XJ24‐24A and cause a quite significant rearrangement of the whole region. We performed analysis of YCR28, the largest open reading frame we found in both AB972 and XJ24‐24A. YCR28 encodes a putative protein of 512 amino acids with some similarities to yeast allontoate permease. Its disruption does not cause any detectable phenotype on rich medium or on allantoate medium, while we observed a strain‐dependent effect on senstivity to amino acid balance and to 3‐aminotriazole, when cells were grown in synthetic medium.
Stuctural and putative regulatory sequences of Kluyveromyces ribosomal protein genes
Authors: Bergkamp‐Steffens G. Karin , Hoekstra Ruurdtje , Planta Rudi J.
Published: May 2005

The transcription of the majority of the ribosomal protein (rp) genes of Saccharmoyces cerevisiae is activated by cis‐acting elements, designated RPG boxes, which specifically bind the multifunctional protein RAPI in vitro.

To investigate to what extent this global system of tanscription regulation has been conserved, we have isolated a number of rp genes of the related yeast species Kluyveromyces lactis and Kluyveromyces marxianus, whose counterparts in Saccharomyces are controlled by RAPI. The coding regions of these genes showed a sequence similarity of about 90% when compared to their Saccharomyces counterparts. In contrast, little or no sequence similarity was found between the upstream regions and the intervening sequences of Kluyveromyces and Saccharomyces homologs. However, the occurrence and the position of the introns is conserved. The sequence data also show that the physical linkage that exists in S. cerevisiae between the rp genes encoding RP59 (CRY1), S24 and L46 is conserved in Kluyveromyces. Northern analysis demonstrated that each of the isolated Kluyveromyces genes is transcriptionally active.

By sequence comparison we idetified a number of conseerved sequences in the upstream region of each of the Kluyveromyces rp genes, which we designated the X, Z and RPGK boxes. The last one is highly similar, though not identical, to the S. cerevisiae RPG box.

Functional analysis of the intergenic region between the genes encoding Kluyveromyces ribosomal proteins S24 and L46 showed that the RPGK box (+Z box) functions as a transcriptional activator, while the X box acts as a trascriptional repressor. Band‐shift assays confirmed the existence of a RAP1‐like protein in Kluyveromyces that binds to the RPGK box but not to the S. cerevisiae RPG box. In contrast, S. cerevisiae RAP1 did recognize the RPGK box.
Parameters affecting the frequencies of transformation and co‐transfromation with synthetic oligonucleotides in yeast
Authors: Yamamoto Tetsuro , Moerschell Richard P. , Wakem Paul , Ferguson David , Sherman Fred
Published: May 2005

Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially fuctional iso‐1‐cytochrome c. Aproximately 3 × 104 transformanrs, constituting 0·1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogenous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double‐strand plasmid, was efficient in a rad52− strain, ssuggesting that the pathway for transformation with oligonucleotides is different from that with linearized double‐strand plasmid. We describe a procedure of co‐transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amono acid alteration elsewhere in the iso‐1‐cytochrome c molecule; approximately 20% of the transformants obtained by co‐transformation contained these desired second alterations.
Structure and expression of the ABF1‐regulated ribosomal protein S33 gene in Kluyveromyces
Authors: Hoekstra Ruurdtje , Ferreira Pedro Moradas , Bootsman Theo C. , Mager Willem H. , Plnata Rudi J.
Published: May 2005

The abundant multifuctional protein ABF1 of Saccharomyces cerevisiae binds to the upstream region of several genes, including some ribosomal protein genes like the one encoding protein S33. Deletion of th ABF1‐binding sequence lowers the transcription of these genes three‐ to more than ten‐fold.

We have isolated the S33 genes of two related yeast species. Kluyveromyces lactis and Kluveromyces marxianus. Comparison of the nucleotide sequences of these S33 genes with their counterpart form S. Cerevisiae shows a strong sequence similarity covering the whole of the coding regions. In contrast, little or no sequence similarly is found in the 5′‐flanking regions of the three genes. Also the trailer regions differ considerably in both length and sequence from one species to another.

An ABF1‐binding site is present in the upstream region of the S33 gene of K. marxianus. Retardation analyses showed that this sequences is able to bind a protein present in Kluyveromyces cells with a molecular mass somewhat lower than that of S. cerevisiae ABF1. Functional analyses, using a β‐glucuronidase reporter system, showed that the ABF1‐binding site is indeed involved in transcription activation of the K. marxianus S33 gene in Kluyveromyces DNA and Northern blots did not show a signal.

These results indicate that S. cerevisiae and Kluyveromyces contain functionally related but structurally dissimilar ABF1‐type proteins.
Mapping the putative RNA helicase genes by sequence overlaping
Authors: Chang Tien‐Hsien , Baum Bobby
Published: May 2005

An ‘electroinic’ gene mapping procedure based on computer‐aided search for overlapping gene sequences was used to identify adjacent genes and localize several putative RNA helicase genes to different chromosomes. PRP28 and AMD1 genes map to the right arm of chromosome IV netxt to sup2, which encodes a tyrsine tRNA PRP16, previouslymapped to chromosome XI, is tightly linked to MRP − L20. PRP22 is adjacent to PRE1, whose chromosomal location is currently unknown. The utility of this aproach in yeast gene mapping is evaluated.
XI. Yeast sequencing reports. The sequence of a 12kb fragment on the left arm of yeast chromosome XI reveals five new open reading frames, including a zinc finger protein and a homolog of the UDP‐glucose pyrophosphorylase from potato
Authors: Purnelle Bénédicte , Skala Jacek , Van Dyack Luc , Goffeau André
Published: May 2005

We report the sequence of a 12 299 bp DNA fragment from the left arm of Saccharomyces cerevisiae chromosome XI. This fragment is located between the genetic loci mif2 and mak11. We have detected five new open reading frames (ORFs) longer than 300 bp provisionally called YKL248 to YKL252. One of them, YKL248 encodes a homolog of the UDP‐glucose pyrophosphorylase from potato. The product of YKL251 contains the consensus for zinc binding proteins, similar to those of a number of fungal transcriptional activators. The three other ORFs do not show significant homology to known protein.
The allantoin and uracil permease gene sequences of Saccharomyces cerevisiae are nearly identical
Authors: Yoo Hyang Sook , Cunningham Thomas S. , Cooper Terrance G.
Published: May 2005

We have determined the structure of the allantoin permease (DAL4) gene of Saccharomyces cerevisiae. The gene patatively encodes a hydrophobic protein with a Mr of 71 755. It possesses the alternating hydrophobic–hydrophilic regions similar to those found in many other integral membrane proteins. The most striking feature of the allantoin permease component encoded by DAL4 is its striking similarity to the uracil permease component encoded by FUR4 Although data available indicate that these proteins do not share any overlap of function, their predicted protein sequences are 68% identical, 81% similar, and their DNA sequences are 70% identical. The upstream regulatory region of DAL4 contains all lof the characterized cis‐acting elements previously reported for inducible allantoin pathway genes: six sequences homologous to UAS NTR, the element responsible for nitrogen catabolite repression‐sensitive activation of allantoin pathway gene expression, and two sequences homologous to the cis‐acting element responsible for inducere‐responsiveness of the allantoin pathway genes, UIS. The finding of these homologous sequences predicted to exist on the basis of DAL4's expression characteristics, supports and strengthens the suggestion that these elements mediate the functions we have we have previously ascribed to them.
DNA transformations of Candida tropicalis with replicating and integrative vectors
Authors: Sanglard Dominiqaue , Fiechter Armin
Published: May 2005

The alkane‐assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV‐mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK 16, that was developed for the transformation of C. albicans and caries an ADE2 gene marker and a Canadida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK 16 under non‐selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK 16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.
V. Yeast mapping reports. Genetic and physical mapping of the WBP1 locus close to CENV
Authors: Heesen Stephan Te , Aebi Markus
Published: May 2005

The WBP1 locus, encoding an essential component of the N‐oligosaccharyl transferase, was mapped both genetically and physically. The gene is located on chromosome V between CENV and gcn4. The distance from CENV sequences is 2 kb.
Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae
Authors: Teunissen Aloys W. R. H. , Van Den Berg Johan A. , Steensma H. Yde
Published: May 2005

The genetics of flocculation in the yeast Saccharomyces cerevisiae are poorly understood despite the importance of this property for strains used in industry. To be able to study the regulation of flocculation in yeast, one of the genes involved, FLO1, has been partially cloned. The identity of the gene was confirmed by the non‐flocculent phenotype of cells in which the C‐terminal part of the gene had been replaced by the URA3 gene. Southern blots and genetic crosses showed that the URA3 gene had integrated at the expected position on chromosome I. A region of approximately 2 kb in the middle of the FLO1 gene was consistently deleted during propagation in Escherichia coli and could not be isolated. Plasmids containing the incomplete gene, however, were still able to cause weak flocculation in a nonflocculent strain. The 3′ end of the FLO1 gene was localized at approximately 24 kb from the right end of chromosome I, 20 kb centromere‐proximal to PHO11. Most of the newly isolated chromosome I sequences also hybridized to chromosome VIII DNA, thus extending the homology between the right end of chromosome I and chromosome VIII to approximately 28 kb.
A yeast antiviral protein, SKI8, shares a repeated amino acid sequence pattern with β‐subunits of G proteins and several other proteins
Authors: Matsumoto Yutaka , Sarkar Gobinda , Sommer Steve S. , Wickner Reed B.
Published: May 2005

SKI8 is a yeast antiviral gene, essential for controlling the propagation of M double‐stranded RNA (dsRNA) and thus for preventing virus‐induced cytopathology. Our DNA sequence of SKI8 shows that it encodes a 397 amino acid protein containing two copies of a 31 amino acid repeat pattern first identified in mammalian β‐transducin and Cdc4p of yeast. There are also four copies of this repeat in yeast Mak11p, necessary for M dsRNA propagation, and three copies in the putative product of the Dictyostelium AAC3 gene. Analysis of 36 cases of the repeat unit shows they have a consensus predicted structure: N–helix–sheet–turn–sheet–turn–sheet–helix–C.
Yeast flocculation: Flocculation onset and receptor availability
Authors: Stratford Malcolm
Published: May 2005

Flocculent strains of brewing yeast grow and ferment as single cells and flocculate in the stationary phase of growth. The switch from single‐celled yeast growth to multi‐celled aggregation, flocculation onset, is of critical importance to the brewing industry. Yeast flocculation involves adhesion of surface‐lectins on flocculent cells to carbohydrate receptors on neighbouring cell‐walls. The presence of carbohydrate receptors, outer‐chain mannan side‐branches, was monitored throughout growth of flocculent and non‐flocculent strains of Saccharomyces cerevisiae, with particular attention to the growth phases where flocculation is normally developed. Receptors were probed by coflocculation with flocculent strains and by aggregation with concanavalin A, a lectin shown to use the same receptors as flocculation.

While considerable variation was found in coflocculation and concanavalin A aggregation between strains, little or no change in receptor availability was found throughout the growth of all yeast strains. Yeast cells could easily be coflocculated at any growth stage. It was concluded that receptor availability is not involved in the process of flocculation onset.
The VPH2 gene encodes a 25 kDa protein required for activity of the yeast vacuolar H+‐ATPase
Authors: Bachhawat Anand K. , Manolson Morris F. , Murdock Deborah G. , Garman J. David , Jones Elizabeth W.
Published: May 2005

Strains bearing the vph2 mutation are defective in vacuolar acidification. The VPH2 gene was isolated from a genomic DNA library by complementation of the zinc‐sensitive phenotype of the mutant. Deletion analysis localized the complementing activity to a 1·2 kb DNA fragment. Sequence analysis of this fragment revealed the presence of a single open reading frame that encoded a protein of 215 amino acids. Computer analysis indicated that the protein, which has a predicted molecular mass of 25 286 Daltons, has two distinct membrane‐spanning domains. Biochemical studies indicated that strains bearing the vph2 mutation have greatly reduced levels of vacuolar proton pumping and ATPase activity and that the nucleotide binding subunits of the multimeric vacuolar H+‐ATPase failed to be correctly targeted to the vacuolar membrane. The vph2 mutant fails to grow on YEP glycerol medium and on media containing 100 mM‐CaCl2 or 4 mM‐ZnCl2 or buffered to pH 7·5, a phenotype observed in strains carrying deletions in the genes encoding several vacuolar H+‐ATPase subunits. The VPH2 gene is identical to the VMA12 gene (T. Stevens and Y. Anraku, personal communication).
Peroxisomal amine oxidase of Hansenula polymorpha does not require its SRL‐containing C‐terminal sequence for targeting
Authors: Faber Klaas Nico , Haima Peter , De Hoop Meltsje Janke , Harder Wim , Veenhuis Marten , Ab Geert
Published: May 2005

Amine oxidase (AMO) is a peroxisomal matrix protein of Hansenula polymorpha, which is induced during growth of the yeast in media containing primary amines as a sole nitrogen source. The deduced amino acid sequence of the protein contains an SRL sequence at nine amino acids from the C‐terminus. In this study, we have examined the possible role of the SRL motif in sorting of AMO to peroxisomes by mutating the corresponding gene sequence. For this purpose, we have developed a DNA construct that is specifically integrated into the AMO locus of the H. polymorpha genome, placing the mutant gene under the control of the endogenous AMO promoter and eliminating expression of the wild‐type gene. Analysis of a stable transformant, containing the desired gene configuration, showed that mutation of the C‐terminal sequence neither interfered with correct targeting of the protein into the peroxisome nor displayed significant effects on its activity. From this, it was concluded that the SRL‐containing C‐terminus is not essential for peroxisomal targeting of AMO in H. polymorpha.
Yeast flocculation: Lectin synthesis and activation
Authors: Stratford Malcolm , Carter Andrew T.
Published: May 2005

Yeast flocculation involves binding of surface lectins to carbohydrate receptors on neighbouring cell walls. Brewing strains of Saccharomyces cerevisiae normally become flocculent in the stationary phase of growth. This paper presents evidence that lectins are synthesized in exponential phase, inserted into the cell wall, and activated later at the time of flocculation onset.

Cycloheximide failed to prevent flocculation unless it was added in early growth; with later additions progressively larger degrees of flocculation occurred. Flocculation onset was delayed by cycloheximide but was otherwise cycloheximide insensitive. Preflocculent cells could be artificially activated to full flocculation by heat. Artificial activation of samples from growing yeast cultures confirmed the progressive synthesis of lectins throughout exponential growth. Pronase E treatment of whole cells prior to heating prevented any activation of flocculation.

It was concluded that lectins were synthesized continuously from an early stage of growth and rapidly inserted into the cell wall (accessible by pronase E), where they remained inactive for up to 14 h, before being activated at flocculation onset by an as‐yet unknown mechanism. It was found that lectin synthesis and activation occurred in all brewing strains tested.
Carbon source induces growth of stationary phase yeast cells, independent of carbon source metabolism
Authors: Granot David , Snyder Michael
Published: May 2005

Nutrients regulate the proliferation of many eukaryotic cells: in the absence of sufficient nutrients vegetatively growing cells will enter stationary (G0 like) phase; in the presence of sufficient nutrients non‐proliferative cells will begin growth. Previously we have shown that glucose is the critical nutrient which stimulates a variety of growth‐related events in the yeast Saccharomyces cerevisiae (Granot and Snyder, 1991). This paper describes six new aspects of the induction of cell growth events by nutrients in S. cerevisiae. First, all carbon sources tested, both fermentable and non‐fermentable, induce growth‐related events in stationary phase cells, suggesting that the carbon source is the critical nutrient which stimulates growth. Second, the continuous presence of glucose is not necessary for the induction of growth events, but rather a short ‘pulse’ of glucose followed by an incubation period in water will induce growth events. Third, growth stimulation by glucose occurs in the absence of the SNF3 high affinity glucose transporter. Fourth, growth stimulation occurs independent of carbon source phosphorylation and carbon source metabolism. Fifth, growth induction by carbon source does not require protein synthesis or extracellular calcium. Sixth, following stimulation by carbon source, the cells remain induced for more than 2 h after removal of the carbon source. We suggest a general model in which different carbon sources act as signals to induce the earliest growth events during or following its entry into the cell and that these growth events do not depend upon metabolism of the carbon source.
The NUM1 yeast gene: Length polymorphism and physiological aspects of mutant phenotype
Authors: Revardel Emmanuelle , Aigle Michel
Published: May 2005

We have isolated a mutant (rvs272) of the yeast (Saccharomyces cerevisiae) that displays an altered phenotype in stationary phase. It shows a high proportion of large‐budded cells with two non‐segregated nuclei staying in the mother cell. This phenotype is also expressed in various conditions when cells are synchronized, energy depleted or treated with the antimitotic drug benomyl. The RVS272 gene has been identified as the NUM1 gene already described. This gene presents a 192 bp tandemly repeated motif and we show that the number of repeats can vary from 1 to about 24 among different strains, without apparently affecting the function of the encoded protein. We suggest that this protein could be involved in polymerization catalysis and/or stabilization of microtubules.
The yal017 gene on the left arm of chromosome I of Saccharomyces cerevisiae encodes a putative serine/threonine protein kinase
Authors: Clark Michael W. , Zhong Wu Wei , Ouellette B. F. Francis , Bussey Howard , Keng Teresa , Storms Reginald K. , Barton Arnold , Kaback David B.
Published: May 2005

The DNA sequence of a region between the LTE1 and CYS3 genes on the left arm of chromosome I from Saccharomyces cerevisiae contains an open reading frame (ORF), YAL017, corresponding to the 5·0 kb FUN31 (Function Unknown Now) transcribed region. The predicted protein from this ORF contains 1358 amino acid residues with a molecular weight of 152 531, and an identifiable serine/threonine protein kinase catalytic domain. When compared with other yeast protein kinases, the Ya1017p kinase most resembles the SNF1 serine/threonine protein kinase which is involved in regulating sucrose fermentation genes. The Ya1017p kinase shows highest amino acid identities with two mammalian carcinoma‐related serine/threonine protein kinases; PIM‐1, which shows induced expression in T‐cell lymphomas; and p78A1, whose expression is lost in human pancreatic carcinomas. Gene disruption of YAL017 indicates that it is non‐essential for growth on glucose.
Identification of a gene encoding a novel zinc finger protein in Saccharomyces cerevisiae
Authors: Breitwieser Wolfgang , Schuster Tillman , Price Clive
Published: May 2005

In the course of a comprehensive genomic screen for cell cycle regulated genes in the yeast Saccharomyces cerevisiae (Price et al., 1991) we identified and characterized a transcriptional unit encoding a putative zinc finger protein named FZF1 (EMBL accession number: X67787). This gene encodes a protein containing five zinc fingers of the Cys2His2 class, three of which are positioned in tandem at the N‐terminus. The fourth and fifth finger follow after an interruption of 61 and 66 amino acids, respectively. While FZF1 is constitutively expressed at a very low level, its deletion is not essential for growth. Its similarity with known transcription factors, however, suggests that the FZF1 gene product may serve as a transcription factor in yeast.
Alteration of cell population structure due to cell lysis in Saccharomyces cerevisiae cells overexpressing the GAL4 gene
Authors: Martegani Enzo , Brambilla Luca , Porro Danilo , Ranzi Bianca Maria , Alberghina Lilia
Published: May 2005

Transformed Saccharomyces cerevisiae cells overexpressing the Escherichia coli LacZ gene and the transcriptional activator GAL4, release in the external medium a fraction (from 2 to 10%) of the total β‐galactosidase activity (Porro et al., 1992b). It is known that this abnormal release of a cytoplasmic protein is related to a partial cell lysis of the yeast population, which is likely to be caused by the overexpression of the transcriptional activator GAL4.

In the present paper we have characterized the GAL4‐induced cell lysis phenomenon. The expression of the GAL4 gene causes morphological modifications and alteration of the cell size distribution. The cell lysis is independent of the expression of the heterologous LacZ gene and occurs in a specific subpopulation of cells (the parent cells) independently of the genealogical age, growth phase conditions and cell cycle progression. Lysis is preceded by a loss of the plasma membrane integrity as indicated by the uptake of ethidium bromide in unfixed cells.

Computer analysis of simulated protein distributions indicates that cell lysis takes place in a sizeable aliquot (about 50%) of the parent cells, therefore profoundly altering the age structure of the population.
Known heat‐shock proteins are not responsible for stress‐induced rapid degradation of ribosomal protein mRNAs in yeast
Authors: Galego Lisete , Barahona Isabel , Alves Ana‐Paula , Rodrigues‐Pousada Claudina , Vreken Peter , Raué Hendrik A. , Planta Rudi J.
Published: May 2005

We have previously shown that the heat‐induced enhanced decay of yeast mRNAs encoding ribosomal proteins (rp‐mRNAs) requires ongoing transcription during the heat treatment [Herruer et al. (1988) Nucl. Acids Res. 16, 7917]. In order to determine whether this requirement reflects the need for heat‐shock protein (hsp), we analysed the effect of heat shock on rp‐mRNA levels in several yeast strains in which each of the heat‐shock genes encoding hsp26, hsp35 or hsp83 had been individually disrupted. In all three strains we still observed increased degradation of rp‐mRNAs immediately after the temperature shift, demonstrating that hsp26, hsp35 and hsp83 are not required for this effect. Accelerated turnover of rp‐mRNA was also found to occur upon raising the growth temperature of a mutant strain that contains a disruption of the gene specifying the heat‐shock transcription factor and in wild‐type yeast cells treated with canavanine, an arginine analogue that will be incorporated into all known hsps and that is known to cause misfolding of the polypeptide chain. Latter observation suggests that enhanced rp‐mRNA decay is a more general stress‐related phenomenon. Taken together, these data strongly indicate that the trans‐acting factor required for the increase in the rate of degradation of rp‐mRNAs upon stress is not one of the known yeast hsps.
Molecular analysis of HEM6 (HEM12) in Saccharomyces cerevisiae, the gene for uroporphyrinogen decarboxylase
Authors: Diflumeri Celestino , Larocque Robert , Keng Teresa
Published: May 2005

HEM6 (HEM12) in Saccharomyces cerevisiae encodes uroporphyrinogen decarboxylase, the fifth enzyme in the heme biosynthetic pathway. The HEM6 (HEM12) gene was cloned by complementation of heme auxotrophy of a hem6 mutant. Sequence analysis revealed an open reading frame of 1086 nucleotides. The predicted amino acid sequence of HEM6 (HEM12) shows extensive homology to those reported for uroporphyrinogen decarboxylase from mammalian sources. Expression of HEM6 (HEM12) was investigated and was found to increase two‐fold in a non‐fermentable carbon source. However, HEM6 (HEM12) transcription was unaffected by heme or by intermediates in the heme biosynthetic pathway. In addition, HEM6 (HEM12) expression is not regulated by the transcriptional activator complex HAP2–3–4, as has been shown for some genes encoding heme biosynthetic enzymes.
Cloning and sequencing of the URA3 gene of Kluyveromyces marxianus CBS 6556
Authors: Bergkamp Ronald J. M. , Geerse Ruud H. , Planta Rudi J. , Verbakel John M. A.
Published: May 2005

The URA3 gene, coding for orotidine‐5′‐phosphate decarboxylase, from Kluyveromyces marxianus CBS 6556, was isolated from a genomic DNA library. The K. marxianus URA3 gene encodes a protein of 267 amino acids with a calculated molecular weight of 29·3 kDa. Comparison of the K. marxianus protein with the corresponding enzymes of Saccharomyces cerevisiae and Kluyveromyces lactis showed amino acid sequence identifies of 81% and 88%, respectively. Using contour‐clamped homogeneous electric field gel electrophoresis, the genomic copy was found to be located on chromosome VI. We have used the cloned gene for the construction of a K. marxianus leu2 mutant. This mutant contains no heterologous sequences, which is essential to make it acceptable for application in the food industry.
Isolation of single yeast cells by optical trapping
Authors: Grimbergen Jos A. , Visscher Koen , De Mesquita Daniel S. Gomes , Brakenhoff G. J.
Published: May 2005

Individual yeast cells can be successfully isolated and recultured on plates with a new isolation method making use of optical trapping with infrared laser light. The cells can be selected on morphological criteria by high resolution microscopy. The isolation device is constructed from two coverslips separated by spacers, in which selected cells are transferred to a plastic capillary, using the optical trap. To test the procedure, selection experiments were done with a mixture of two Saccharomyces cerevisiae strains, distinguishable both in fluorescence microscopy and on agar plates. These experiments showed that only selected cells were isolated, and close to 100% of the isolated stationary‐phase cells formed colonies on agar plates, indicating a high recovery. A lower recovery was obtained with exponential‐phase cells, possibly because of a higher sensitivity to laser irradiation. Applications for this method may include the isolation of mutants with altered morphology and the isolation of subpopulations of yeast cultures, for their separate investigation or for the initiation of pure cultures.
Different signals control the activation of glycolysis in the yeast Saccharomyces cerevisiae
Authors: Boles Eckhard , Zimmermann Friedrich K. , Heinisch Jürgen
Published: May 2005

The glycolytic pathway in Saccharomyces cerevisiae is activated by fermentable sugars at several steps. Mutants with deletions of genes coding for enzymes of the upper part of glycolysis were used to characterize the triggering mechanisms. Synthesis of fructose‐2,6‐bisphophate is catalysed by two 6‐phosphofructo‐2‐kinase isoenzymes, one of which is activated by fermentable sugars while synthesis of the second enzyme is induced (Kretschmer and Fraenkel, 1991). Increase in the level of fructose‐2, 6‐bisphosphate is demonstrated to depend on an internal metabolite upstream of the phosphoglucose isomerase reaction. The signalling process correlates with distinct temporal changes in the concentration of glucose‐6‐phosphate but not with its absolute level, indicating an adaptational mechanism. It is independent of the uptake and phosphorylation systems used by different sugars. Interestingly, this increase, although delayed, could also be observed in strains lacking the rapid cAMP increase after sugar addition which is thought to be responsible for the activating process. Synthesis of glucose‐6‐P and fructose‐6‐P is needed for the complete induction of pyruvate kinase and inactivation of fructose‐1,6‐bisphosphatase. On the other hand, induction of pyruvate decarboxylase depends mainly on a signal in the lower part of glycolysis.
The multifunctional regulatory proteins ABF1 and CPF1 are involved in the formation of a nuclease‐hypersensitive region in the promoter of the QCR8 gene
Authors: De Winde Johannes H. , van Leeuwen Hans C. , Grivell Leslie A.
Published: May 2005

The abundant DNA‐binding proteins ABF1 and CPF1 are members of a family of global regulators with diverse chromosomal functions in the yeast Saccharomyces cerevisiae. Recent evidence suggests that these protein factors may be involved in establishing and maintaining well‐defined chromatin structures in promoter regions and other genetic elements. We have investigated the involvement of ABF1 and CPF1 in chromatin organization at the QCR8 gene, encoding subunit VIII of the mitochondrial ubiquinol‐cytochrome c oxidoreductase. The promoter region of the QCR8 gene contains overlapping binding sites for ABF1 and CPF1. Nucleosome positioning studies indicate that the QCR8 gene is associated with a phased array of nucleosomes under both catabolite‐repressed and derepressed growth conditions. Analysis of binding site mutants reveals that both ABF1 and CPF1 are involved in maintaining a nuclease‐hypersensitive region in the QCR8 promoter. The chromatin structure at QCR8 during steady‐state growth is, however, mainly dependent on binding of ABF1 to the promoter region. Implications of these findings for the role played by ABF1 and CPF1 in the regulation of mitochondrial biogenesis and other processes important for cell growth and division will be discussed.
Cloning and characterization of the SEC18 gene from Candida albicans
Authors: Nieto Almudena , Sentandreu Rafael , Agudo Lucas Del Castillo , Sanz Pascual
Published: May 2005

The SEC18 gene product is required for protein transport at different stages in the Saccharomyces cerevisiae secretory pathway. The homologous SEC18 gene from Candida albicans has been cloned by complementation of a sec18‐1 S. cerevisiae thermosensitive mutant using a C. albicans genomic library in YRp7. Sequence analysis of the gene revealed a 2382‐bp open reading frame which coded for a protein of 88 926 kDa. By an in vitro transcription–translation coupled reaction of the C. albicans SEC18 gene, a protein of approximately 85 kDa was obtained. Hydrophobicity analysis of the protein did not show any predicted signal sequence nor transmembrane anchor domain. These results and the fact that glycosylation was absent in the protein indicated that C. albicans Sec18p did not enter in the secretory pathway. The alignment of the amino acid sequence revealed that the SEC18 gene from C. albicans was homologous to the SEC18 from S. cerevisiae (50% amino acid identity) and to the gene that coded the N‐ethylmaleimide‐sensitive factor (NSF) protein (43% amino acid identity). Moreover, the C. albicans Sec18p also showed the putative ATP binding site present in S. cerevisiae Sec18p and in NSF.
Isolation and DNA sequence of the STE14 gene encoding farnesyl cysteine: Carboxyl methyltransferase
Authors: Ashby Matthew N. , Errada Patrick R. , Boyartchuk Victor L. , Rine Jasper
Published: May 2005

We isolated a mutant defective in C‐terminal farnesyl cysteine:carboxyl methyltransferase activity from a screen for mutations causing a‐specific sterility. A genomic fragment was cloned from a yeast multi‐copy library that restored mating. Both the cloned gene and the sterile mutation were allelic to the STE14 gene. A ste14‐complementing 2·17 kb BamHI fragment subclone was sequenced and found to encode a 239 amino acid protein with a molecular weight of 27,887 Daltons. The hydrophobicity profile of the methyltransferase reveals the presence of at least five potential transmembrane domains. In comparisons of the C‐terminal methyltransferase amino acid sequence with those in the PIR and Swiss protein databases, no significantly similar sequences were found nor were conserved regions from other methyltransferases present.
KTR2: A new member of the KRE2 mannosyltransferase gene family
Authors: Lussier Marc , Camirand Anne , Sdicu Anne‐Marie , Bussey Howard
Published: May 2005

The KTR2 gene from Saccharomyces cerevisiae was identified by polymerase chain reaction amplification of genomic DNA using primers derived from regions of high homology between the products of three yeast genes, KRE2, YUR1 and KTR1. The product encoded by the KTR2 gene is a predicted type II membrane protein of 425 amino acid residues with a short cytoplasmic N‐terminus, a membrane‐spanning region and a large lumenal domain containing four potential N‐glycosylation sites. Ktr2p has 58% identity with Yur1p, 39% with Ktr1p and 34% with Kre2p. One member of this gene family, KRE2 (also known as MNT1; Häusler and Robbins, 1992), encodes an α‐1,2 mannosyltransferase which adds the third mannose onto O‐linked glycoprotein side‐chains (Häusler et al., 1992). In contrast to KRE2 null mutants, which produce shortened (two‐mannose) chains, mutants harboring a KTR2 gene disruption synthesize O‐linked chains with the wild‐type patterns of five mannose residues. A null mutation in KTR2 leads to partial resistance to killer toxin and hints that KTR2, which encodes a putative mannosyltransferase, is involved in extracellular matrix assembly.
Regulation of the amino acid permeases in nitrogen‐limited continuous cultures of the yeast Saccharomyces cerevisiae
Authors: Olivera Hiram , González Alicia , Peña Antonio
Published: May 2005

In the yeast Saccharomyces cerevisiae, there is a general amino acid permease, regulated by nitrogen catabolite repression, and several specific permeases whose nitrogen regulation is not well understood. In this study, we used continuous cultures to analyse the effect of nitrogen limitation and pH on the activity of general and several specific amino acid permeases. General permease activity was maximal in severe nitrogen limitation and diminished 400‐fold in cells grown under nitrogen excess. For the specific permeases, the maximal uptake activity was found between mild limitation and nitrogen excess, while very small activity was detected under strict limitation. These results indicate that the nitrogen regulation of the general and the specific amino acid carriers is coordinated in such a way that no redundancy exists in amino acid transport. The regulation of the specific permeases was similar to that found for a system with anabolic function in nitrogen metabolism.

All of these permeases are supposed to work through a proton symport mechanism, and thus rely on pH gradients to carry out their function. We studied the effect of pH on the kinetic constants of the general permease. Our results show that the effect of pH on the Km was different for acidic, neutral and basic amino acids, while the effect on Vmax was independent of the electrical charge of the amino acids.
PFK2, ISP42, ERG2 and RAD14 are located on the right arm of chromosome XIII
Authors: Heinisch Jûrgen J.
Published: May 2005

An 11 kb yeast DNA insertion isolated from a genomic library by complementation of a phosphofructokinase‐deficient strain was used as a source for a partial sequence analysis. Four genes were shown to reside on this fragment, namely PFK2, ISP42, ERG2 and RAD14. PFK2 was mapped previously to the right arm of chromosome XIII, locating the latter three genes to the same chromosome.
The RHO4a and NUD1 genes on Saccharomyces cerevisiae chromosome XI
Authors: Van Vliet‐Reedijk Joke C. , Planta Rudi J.
Published: May 2005

Sequence analysis of a 4 kb fragment from the right arm of Saccharomyces cerevisiae chromosome XI, in combination with Northern hybridization experiments revealed the presence of two genes, designated RHO4a and NUD1. The first gene encodes a 32 kDa protein showing significant sequence similarity with members of the ras family. Its 3′‐terminal sequence is virtually identical to a sequence published previously as the RHO4 gene [Matsui and Toh‐e, Gene 114 (1992), 43–49], which, however, appears to start at an internal ATG codon. The RHO4a sequence also overlaps the 5′‐terminal sequence of the RNC1 gene [Chow et al., Nucl. Acids Res. 20 (1992), 5215–5221] proposed to encode the yeast yNucR endo/exonuclease. The remainder of this RNC1 gene overlaps with the 5′‐end of the NUD1 gene. However, the RNC1 sequence lacks a portion of 276 bp that in our fragment is part of the intergenic region separating RHO4a and NUD1. From these results we conclude that the proposed RNC1 gene is the result of a cloning artefact and that the yNucR protein is instead encoded by the NUD1 gene.
Nucleotide sequence analysis of the 5·8S rDNA and adjacent ITS2 region of Candida albicans and related species
Authors: Lott Timothy J. , Kuykendall Randall J. , Reiss Errol
Published: May 2005

We have determined the nucleotide sequence for the DNA encoding the 5·8S RNAs and downstream internal transcribed spacer (ITS2) regions for Candida albicans and the taxonomically related species C. parapsilosis, C. tropicalis, C. glabrata and C. krusei. Phylogenetic analysis of all known fungal 5·8S RNA sequences revealed a close relationship between C. tropicalis and C. parapsilosis, and to a lesser extent C. albicans within the yeast‐like fungi. This group can itself be delineated from predominantly filamentous species. The more distal relationships between Candida (torulopsis) glabrata and C. krusei support previous findings based on small (18S) ribosomal RNA sequence analysis, suggesting a greater degree of evolutionary divergence of these species from the C. albicans group. Among strains of C. albicans we observed conservation of the ITS2 region of the nucleotide level. Conservation was also observed for a more limited number of C. parapsilosis strains. Although the 3′ region of the ITS spacer was species specific, sequence homology was observed in the 5′ end within the albicans/parapsilosis/tropicalis group. Our findings suggest a rapid approach to species identification through the use of non‐conserved regions flanked by highly conserved, functional domains.
The electrophoretic karyotype of two strains of Candida albicans by transverse alternate field electrophoresis reveals higher number of chromosomes ranging from 1 to 3·5 Mb
Authors: Allegrucci Massimo , Lanfaloni Luisa , Bietta Carla , Spaccapelo Roberta , Fioretti Maria C. , Bistoni Francesco
Published: May 2005

The advent of the powerful electrophoretic technique, pulsed field gel electrophoresis, first developed on the yeast Saccharomyces cerevisiæ, has brought a vital impulse to the genetic study on the opportunistic pathogen Candida albicans. We report here on sizing and numbering of Candida chromosomes using transverse alternate field electrophoresis. Our results indicate the occurrence of nine to ten electrophoretic bands (depending on type of Candida strain), that range in approximate size from 1 to 3·5 Mbp, and may account for a higher overall chromosome number, because at least two of these bands appear to be doublets. This number of bands, with smaller size, is considerably higher than previously reported.
Chromatin structure of the yeast FBP1 gene: Transcription‐dependent changes in the regulatory and coding regions
Authors: Del Olmo Marcel Lí , Sogo José M. , Franco Luis , Pérez‐Ortín José E.
Published: May 2005

We have studied the chromatin structure of the Saccharomyces cerevisiae FBP1 gene, which codes for fructose‐1,6‐bisphosphatase. A strong, constitutive, DNase I, micrococcal nuclease and S1 nuclease hypersensitive site is present close to the 3′ end of the coding region. In the repressed state, positioned nucleosomes exist around this site, and subtle changes occur in this nucleosomal organization upon derepression. A DNase I hypersensitive region is located within the promoter between positions −540 and −400 and it extends towards the gene in the derepressed state, leading to an alteration of nucleosomal positioning. Psoralen crosslinking of chromatin, which is used for the first time to study the mobility of restriction fragments from an RNA polymerase II gene, revealed that part of the promoter is nucleosome‐free, in accordance with the results of DNase I digestion. A model is presented that, based on the chromatin structure, puts forward the hypothesis that the promoter UAS is located between − 540 and − 340. Finally, psoralen crosslinking, as well as digestions with micrococcal nuclease or restriction endonucleases suggests that most if not all of the copies of the active FBP1 gene are covered by nucleosomes.
Lysine144 is essential for the catalytic activity of Saccharomyces cerevisiae transaldolase
Authors: Miosga Thomas , Schaaff‐Gerstenschläger Ine , Franken Eva , Zimmermann Friedrich K.
Published: May 2005

Replacement of lysine144 by glutamine in the pentose phosphate pathway enzyme transaldolase of Saccharomyces cerevisiae is associated with the complete loss of activity indicating the essential role in catalysis. Neither histidine nor cysteine is important for catalytic activity as proposed for the Candida utilis enzyme. Also we could not find any evidence for a half‐site character of the enzyme as described for transaldolase of C. utilis. Therefore, the reaction mechanisms for the two enzymes are different.
The 5‐aminoimidazole ribonucleotide‐carboxylase structural gene of the methylotrophic yeast Pichia methanolica: Cloning, sequencing and homology analysis
Authors: Hiep Tran Tuan , Kulikov Vladimir N. , Noskov Vladimir N. , Sizonenko Genadij I. , Chernoff Yuri O. , Pavlov Yuri I.
Published: May 2005

The ADE1 gene of the yeast Pichia methanolica encodes phosphoribosyl‐5‐aminoimidazole‐carboxylase (AIRC, EC 4.1.1.21), which is involved in purine biosynthesis. The gene was cloned by complementation of an ade2 mutation in Saccharomyces cerevisiae and a 3077 nucleotide DNA fragment was sequenced. The sequence possessed a single open reading frame, corresponding to a 543 amino acid sequence. The sequence of this putative protein has been compared to the proteins of homologous genes from S. cerevisiae, Schizosaccharomyces pombe, Escherichia coli, chicken and man. The analysis revealed remarkable homology between yeast AIRCs, while for other proteins homology was limited to defined regions.
Formation of the 3′ end of yeast mitochondrial mRNAs occurs by site‐specific cleavage two bases downstream of a conserved dodecamer sequence
Authors: Hofmann Ted Joseph , Min Jingjuan , Zassenhaus Hans Peter
Published: May 2005

Mitochondrial mRNAs in yeast arise by processing of polygenic primary transcripts at a conserved dodecamer sequence (5′‐AAUAAPyAUUCUU‐3′). Previous results indicated that processing at dodecamer sites interrupted the sequence implying that it functioned primarily as a signal for 3′ end formation of mRNAs. We have determined the precise cleavage site for RNAs processed at the dodecamer sequences associated with the oli1 gene and the ω intron of the 21S rRNA gene. In both cases cleavage occurred two bases downstream of the site. Hydrolysis left the PO4 group attached to the 3′ terminus of the cleavage products. These results demonstrate for the first time that mature mitochondrial mRNAs terminate with an intact dodecamer sequence. In light of the recent identification of a protein complex within mitochondria that binds to RNAs terminating with an intact dodecamer sequence, these results support the idea that the dodecamer sequence functions not only within pre‐mRNAs as a processing site, but within mature mRNAs as well, possibly for the stabilization and/or translation.