bluuebunny/crossref_metadata_2025 huggingface dataset:
Assembly of alcohol oxidase in the cytosol of a peroxisome‐deficient mutant of Hansenula polymorpha—properties of the protein and architecture of the crystals
Authors: Van Der Klei Ida J. , Sulter Grietje J. , Harder Wim , Veenhuis Marten
Published: May 2005

We have studied the expression of alcohol oxidase (AO) in a peroxisome‐deficient mutant strain of Hansenula polymorpha. High levels of octameric, active AO (up to 3·0 U/mg protein) were detected in cells grown at low dilution rates in a glucose‐limited chemostat in the presence of choline as the sole nitrogen source. Monomeric or other intermediate forms of AO were not detected in the mutant strain. This indicated that assembly of the protein into active octameric molecules in the cytosol was as efficient as in wild‐type cells where this process is confined to the peroxisomal matrix. At relatively low rates of expression (less than 1 U/mg protein) AO was localized throughout the cytosol and, surprisingly, was also present inside the nucleus. However, at enhanced levels large crystalloids were formed. Generally one crystalloid was observed per cell, whereas smaller ones were occasionally found in developing buds. Also large crystalloids have been observed inside the nucleus. These crystalloids were not surrounded by a membrane. Based on the morphology of the molecules that constituted these crystalloids and the results of (immuno)cytochemical experiments we conclude that the crystalloids are composed of octameric AO molecules, arranged in a regular lattice, identical to the 3‐dimensional architecture previously described for the crystalline matrix of peroxisomes in methanol‐grown wild type cells of H. polymorpha. Attempts to purify the crystalloids by conventional fractionation methods failed, due to their apparent fragility; however, (immuno)cytochemical experiments revealed that catalase and dihydroxyacetone synthase were also associated with these structures.
Yeast mutants with increased bacterial transposon Tn5 excision
Authors: Gordenin D. A. , Proscyavichus Y. Y. , Malkova A. L. , Trofimova M. V. , Peterzen A.
Published: May 2005

Five complementing recessive mutations that exhibit increased bacterial transposon Tn5 precise excision in yeast Saccharomyces cerevisiae were obtained by ethylmethanesulfonate treatment. One of these mutations (tex1) was submitted to extensive genetic analysis. tex1 is a recessive temperature‐sensitive mutation resulting in a 20–100‐fold increase in Tn5 excision. It also has increased frequencies of ochre mutation reversion, of forward mutation to canavanine resistance, and loss of chromosome III or its right arm. The possible mechanism of tex1 effects is discussed.
Influence of the codon following the initiation codon on the expression of the lacZ gene in Saccharomyces cerevisiae
Authors: Looman A. C. , Laude M. , Stahl U.
Published: May 2005

A set of 32 different codons were introduced in a lacZ experssion vector (pPTK400) immediately 3′ from the AUG initiation codon. Expression of the lacZ gene was determined in Saccharomyces cerevisiae by measuring the amount of β‐galactosidase fusion protein using immuno‐gel electrophoresis. A 5·3‐fold difference in expression was found among the various constructs. It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression. No correlation could be found between the local secondary structure and expression. When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference. This indicates that in yeast, in contrast to Escherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation. Important determinatnts of the optimal context for an initiation codon in yeast therfore must be located mainly 5′ from this codon.
Applications of high efficiency lithium acetate transformation of intact yeast cells using single‐stranded nucleic acids as carrier
Authors: Gietz R. Daniel , Schiestl Robert H.
Published: May 2005

The highly efficient yeast lithium acetate transformation protocol of Schiestl and Gietz (1989) was tested for its applicability to some of the most important need of current yeast molecular biology. The method allows efficient cloning of genes by direct transformation of gene libraries into yeast. When a random gene pool ligation reaction was transformed into yeast, the LEU2, HIS3, URA3, TRP1 and ARG4 genes were found among the primary transformations at a frequency of approximately 0·1%. The RAD4 gene, which is toxic to Escherichia coli, was also identified among the primary transformants of a ligation library at a frequency of 0·18%. Non‐selective transformation using this transformation proctocol was shown to increase the frequency of gene disruption three‐fold. Co‐transformation showed that 30–40% of the transformation‐competent cells take up more than one DNA molecule which can be used to enrich for integration and delection events 30‐ to 60‐fold. Co‐transformation was used in the construction of simultaneous double gene disruptions as well as disrupting both copies of one gene in a diploid which occurred at 2–5% the frequency of the single event.
VII. Yeast sequencing reports. Physical, transcriptional and genetical mapping of a 24 kb DNA fragment located between the PMA1 and ATE1 loci on chromosome VII from Saccharomyces cerevisiae
Authors: Capieaux Etienne , Ulaszewski Stanislaw , Balzi Elisabetta , Goffeau André
Published: May 2005

A physical map of a contiguous DNA fragment of 60 kb, extending from the centromere to TRP5 on the left arm of the chromosome VII of Saccharomyces cerevisiae, strain IL 125‐2B, was established. Within a 31 kb region from PMA1 towards TRP5, a total of 12 transcription products ranging from 0·6 to 3·6 kb were identified in cells grown exponentially on rich medium. Near 87% of the DNA investigated was transcribed and on average one transcript, of 2·3 kb average length, was detected every 2·7 kb of DNA. The physical and genetical distances between the markers CEN7, pma1, leu1, pdr1 and trp5 were compared. A recombination frequency of 1 cM corresponds to an average distance of 3·3 kb between alleles in this region of chromosome VII.
DNA insertions in the ‘silent’ regions of the 2 μm plasmid of Saccharomyces cerevisiae influence plasmid stability
Authors: Bijvoet J. F. M. , Van Der Zanden A. L. , Goosen N. , Brouwer J. , Van De Putte P.
Published: May 2005

The 2 μm plasmid of the yeast Saccharomyces cerevisiae is in principle a suitable vector for expression of foreign genes, due to high copy number and extreme stability. However, the cloning of genes into 2 μm often results in a reduced copy number and/or reduced stability. One reason for this observed instability could be that the inserts in general were made in one of the several open reading frames (ORFs) of the plasmid. Therefore we studied the effect on stability of insertions in the silent regions of 2 μm without interrupting any known essential regions or ORFs. Using the SnaBI site, a yeast‐integrating plasmid (Yip5) was introduced into the region between the ARS and STB locus in two possible orientations. The resulting plasmids could be stably maintained in the cells without the need for complementation by the wild‐type 2 μm plasmid. However, the stability of these plasmids in a cir° host was still one to two orders of magnitude lower (0·2% and 0·8% respectively) as reported for the wild‐type 2 μm (0·01%). Removal of 2 kb of the bacterial sequences from Yip5 did not increase stability. The stability was dependent on the orientation of the insert. We found that in the less stable orientation, transcription originating from the insert was running into the STB region.

DNA inserted in the XmaIII site located outside the ORFs in the REP2/FLP intergenic region influenced both stability and copy number of the plasmid. These effects are strongly dependent on the size of the insert. Insertion of a 2 kb DNA fragment increased the copy number, probably through an effect on FLP expression.
Isolation and quantitation of M‐factor, a diffusible mating factor from the fission yeast Schizosaccharomyces pombe
Authors: Davey John
Published: May 2005

Although there have been several reports demonstrating the existence of mating factors in the fission yeast Schizosaccharomyces pombe it has not been possible to isolate these factors as cell‐free preparations. Such an ability is the first requirement towards a molecular characterization of these factors and here I report the successful isolation of a mating factor from S. pombe. This factor, termed M‐factor, is released by cells of the cellular mating type M (Minus) and induces mating‐specific changes in P‐type cells. A reliable and accurate assay for the quantitation of the M‐factor, based upon changes in cell volume following exposure to the factor, is also described.
Multi‐functional DNA proteins in yeast: The factors GFI and GFII are identical to the ARS‐binding factor ABFI and the centromere‐binding factor CPF1 respectively
Authors: Dorsman J. C. , Gozdzicka‐Jozefiak A. , Van Heeswijk W. C. , Grivell L. A.
Published: May 2005

GFI and GFII are abundant DNA‐binding proteins in the yeast Saccharomyces cerevisiae. Binding sites for GFI conform to the sequence 5′‐RTCRYNNNNNACG‐3′. This consensus can also accommodate the recognition sequence for the ARS1‐binding factor ABFI. Results of retardation‐competition assays, limited proteolysis experiments, molecular weight determinations based on denaturation–renaturation procedures and mobility shift assays of protein–DNA complexes formed in the presence of a monoclonal antibody raised against ABFI suggest strongly that GFI and ABFI are the same protein. Similarly, GFII appears to be identical to the centromere‐binding protein CPFI (alias CP1), since both proteins bind to the CDEI motif of yeast centromeres (5′‐RTCACRTG‐3′) and cannot be detected in a cpf1 disruption mutant yeast strain. In addition, based on denaturation–renaturation studies, both factors appear to have molecular weights in the same range of 53–62 kDa.
III. Yeast sequencing reports. The complete sequence of the unit YCR59, situated between CRY1 and MAT, reveals two long open reading frames, which cover 91% of the 10·1 kb segment
Authors: Jia Yankai , Slonimski Piotr P. , Herbert Christopher J.
Published: May 2005

We have entirely sequenced YCR59, which is a 10·1 kb segment of the right arm of chromosome III, and is a part of the clone E5F from the Newlon collection. The segment contains two long open reading frames (ORFs): YCR591 which starts in the adjacent fragment H9G (situated towards CRY1 and the centromere), and continues with 1833 codons in YCR59. The second ORF YCR592 is 1226 codons long and encoded entirely within YCR59. The two ORFs represent 91% of the total length of the segment. Excellent agreement in both location and length is found between the ORFs YCR591 and YCR592 and the transcripts 86 and 87 respectively in the Yoshikawa and Isono (1990) map of chromosome III. The two ORFs correspond to new genes and show no significant similarity with any known genes.
Cytochrome P450 lanosterol 14α‐demethylase (ERG11) and manganese superoxide dismutase (SOD1) are adjacent genes in Saccharomyces cerevisiae
Authors: Turi Thomas G. , Kalb Vernon F. , Loper John C.
Published: May 2005

DNA sequencing and analysis of genomic DNA using the polymerase chain reaction were used to demonstrate that SOD1 and ERG11 are adjacent genes in Saccharomyces cerevisiae S288c and to establish the correct intergenic sequence of this segment on chromosome VIII.
Activity of promoter mutants of the yeast ribosomal RNA gene with and without the enhancer
Authors: Butlin Mike , Quincey Roger
Published: May 2005

The promoter and enhancer of the rRNA gene of Saccharomyces cerevisiae have been studied using a nuclease S1 protection assay to detect transcripts of an rRNA minigene in transformed yeast. Analysis of 5′ deletion mutants showed that DNA between − 163 bp and − 155 bp was important for promoter activity and that some DNA between − 155 bp and − 145 bp was essential. The importance of DNA far upstream from the initiation site was confirmed by showing that minigene expression was much reduced by linker scanner mutations clustered around − 148 bp, − 133 bp and − 100 bp, and was abolished by mutations clustered around − 118 bp. The enhancer for rRNA biosynthesis increased transcription from all of the five mutated promoters that were tested. The magnitude of the enhancer effects on weakly active promoters was two‐ to three‐fold less than on the wild‐type promoter. Expression of a minor transcript in a 5′ deletion to − 10 bp was substantially reduced by a mutation which altered two base pairs in the core sequence of the promoter‐proximal REB1 binding site.
Functional analysis of a conserved amino‐terminal region of HSP70 by site‐directed mutagenesis
Authors: Nicolet Charles M. , Craig Elizabeth A.
Published: May 2005

Hsp70 proteins have been highly conserved throughout evolution. As a first step in a structure–function analysis of hsp70, we constructed and analysed the consequences of mutations in a portion of the SSA1 gene, a member of the Saccharomyces cerevisiae HSP70 multigene family, that encodes a nearly invariant region near the amino terminus. Analysis of strains expressing SSA1 proteins with alterations at positions 8, 11 and 15 showed that these conserved residues within this region are critical for normal functioning of the protein. SSA1 protein containing either of two changes at position 15 was able to slightly complement the inviability of an ssa1ssa2ssa4 strain, but was inactive in other complementation assays. The other mutant proteins tested were unable to complement any tested phenotype. Effective interallelic complementation of several phenotypes was observed when a mutant protein substituted at position 8 was expressed in the same cell with either of two proteins carrying substitutions at position 15, suggesting that hsp70 acts as a multimer. Evidence from previous studies suggests that hsp70 proteins engage in ATP‐driven cycles of binding and release from peptides. The ability of the mutant proteins to bind ATP and a peptide was tested. The Ssa 1p carrying a substitution at position 8, which inhibits growth of cells carrying wild‐type SSA proteins, showed a defect in release from a peptide relative to wild type. Two mutations, one each at position 8 and 15, resulted in accumulation of phosphorylated isoforms which may be a normal, transient hsp70 intermediate.
The complete sequence of a 7·5 kb region of chromosome III from Saccharomyces cerevisiae that lies between CRY1 and MAT
Authors: Wicksteed Barton L. , Roberts A. Brett , Sagliocco Francis A. , Brown Alistair J. P.
Published: May 2005

We report the sequence of a 7·5 kb region lying between the CRY1 and MAT loci of chromosome III from Saccharomyces cerevisiae. This region lies in the overlap between two major contigs used for the generation of the complete nucleotide sequence of this chromosome. Comparison of this sequence with those reported previously for this overlap [Thierry et al. (1990) Yeast 6, 521; Jia et al. (1991) Yeast 7, 413] reveals 38 nucleotide differences, 45% of which generate changes in the amino acid sequences of the four genes in this region (YCR591, YCR592, YCR521 and YCR522). These differences appear to reflect true sequence polymorphisms between the two yeast strains used to generate the clones used in the sequencing project. Three of the four genes in this region display weak homologies to proteins in the PIR database. Some properties of YCR521 are analogous to those of ribosomal protein genes. However, the functions of all four genes remain obscure.
A genomic sequence of the Schizosaccharomyces pombe 16 kDa vacuolar H+‐ATPase
Authors: Toyama Reiko , Goldstein David J. , Schlegel Richard , Dhar Ravi
Published: May 2005

We have isolated the gene encoding the 16 kDa vacuolar H+‐ATPase from Schizosaccharomyces pombe. On the basis of RNA splicing signals and amino acid sequence homology with other 16 kDa H+‐ATPases, the genomic DNA sequence indicated the 16 kDa protein is encoded by five exons. The C‐terminal 50 amino acids has more than 90% homology with vacuolar H+‐ATPases of mammalian cells.
Expression of recombinant platelet‐derived endothelial cell growth factor in the yeast Saccharomyces cerevisiae
Authors: Finnis Chris , Goodey Andrew , Coutrney Michael , Sleep Darrell
Published: May 2005

A platelet‐derived endothelial cell growth factor cDNA has been cloned, sequenced and expressed using the Saccharomyces cerevesiae PRBI promoter. Soluble recombinant platelet‐derived endothelial cell growth factor constituted 0·5–1·0% of total soluble protein. Yeast soluble protein extracts containing recombinant platelet‐derived endothelial cell growth factor stimulate the growth of calf palmonary artery endothelial cells in vitro.
A putative serine/threonine protein kinase gene on chromosome III of Saccharomyces cerevisiae
Authors: Wilson Cathal , Frontali Laura , Bergantino Elisabetta , Carignani Giovanna , Lanfranchi Gerolamo , Valle Giorgio
Published: May 2005

We have sequenced a gene on chromosome III of Saccharomyces cerevisiae which codes for a putative serine/threonine protein kinase of 726 amino acids (calculated molecular weight 82 kDa). We have called this gene KIN82. The amino acid sequence of KIN82 is most similar to the cyclic nucleotide‐dependent protein kinase subfamily and the protein kinase C subfamily. Gene disruption of KIN82 did not produce any phenotype when tested under a variety of conditons. Reduced stringency hybridizations revealed the presence of another genomic sequence with high homology to the carboxy‐terminal catalytic domain of KIN82.
Inhibition of glycolysis by 2‐deoxygalactose in Saccharomyces cerevisiae
Authors: Lagunas Rosario , Moreno Eulalia
Published: May 2005

The enzymatic steps involved in the inhibition of glycolysis by 2‐deoxygalactose in Saccharomyces cerevisiae have been investigated. Yeast, incubated with 2‐deoxygalactose, accumulates up to 8 mM‐2‐deoxygalactose, 30 mM‐2‐deoxygalactose‐1‐phosphate and 0·25 mM‐UDP‐2‐deoxygalactose and UDP‐2‐dexyglucose. An inverse correlation between 2‐deoxygalactose‐1‐phosphate content and rate of glycolysis has been observed. The intracellular concentration of glycolytic intermediates and related metabolites point to the hexokinase and phosphofructokinase steps as the targets for the inhibition of glycolysis by 2‐deoxygalactose and rule out all other mechanisms that have been proposed to explain this inhibition.
Identification of RAD 16, a yeast excision repair gene homologous to the recombinational repair gene RAD 54 and to the SNF2 gene involved in transcriptioal activation
Authors: Schild David , Mortimer Robert K. , Glassner Brian J. , Carlson Marian , Laurent Brehon C.
Published: May 2005

The RAD54 gene of Saccharomyces cerevisiae is involved in the recombinational repair of DNA damage. The predicted amino acid sequence of the RAD54 protein shows significant homologies with the yeast SNF2 protein, which is required for the transcriptioal activation of a number of diversely regulated genes. These proteins are 31% identical in a 492‐amino acid region that includes presumed nucleotide and Mg2+ binding sites. We noted previously that the SNF2 protein also shares homology with a partial open reading frame (ORF) that was reported with the sequence of an adjacent gene. This ORF also shares homology with the RAD54 protein. To test whether this ORF is involved in transcriptional activation or DNA repair, yeast strains deleted for part of it have been isolated. These strains do not show a Snf‐like phenotyp, but they are UV sensitive. This gene has been identified as RAD 16, a gene involved in the excision repair of DNA damage. Analysis of the rad16 deletion mutations indicates that RAD16 encodes a nonessential function and is not absolutely required for excision repair. Outside the region of homology to RAD54 and SNF2, the predicted RAD16 protein contains a novel cysteine‐rich motif that may bind zinc and that has been found recently in eleven other proteins, including the yeast RAD18 protein. The homologies between RAD16, RAD54 and SNF2 are also shared by several additional, recently isolated yeast and Drosophila genes.
Saccharomyces cerevisiae contains a homolog of human fkbp‐13, a membrane‐associated fk506/rapamycin binding protein
Authors: Partaledis Judith A. , Fleming Mark A. , Harding Matthew W. , Berlin Vivian
Published: May 2005

FKB2 encodes a homolog of human FKBP‐13, a membrane‐associated binding protein for the immunosuppressants FK506 and rapamycin. FKB2 is located on the right arm of chromosome IV and contains an open reading frame of 135 amino acids, of which the first 17 residues comprise a putative hydrophobic leader peptide. Yeast FKBP‐13 is homologous to human FKBP‐13 (52% amino acid identity) and to FKBP‐12, the major cytosolic receptor for FK506. In the alignment of FKBP‐13 and FKBP‐12 sequences, there are 28 invariant residues. Among these conserved residues are those that comprise the drug binding and peptidyl‐prolyl cis‐trans isomerase active site of FKBP‐12. The phylogenetic conservation of the FKBP family suggests that the proteins are involved in a basic cellular function.
Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plusmids
Authors: Kasüske Anette , Wedler Holger , Schulze Steffen , Becher Dietmar
Published: May 2005

Conditions for efficient and quick transformation by electroporation were developed in Candida maltosa. To investigate the efficiency of transformation with integrative as well as with autonomously replicating plasmids, a series of vectors was constructed for homologous transformation of the species. Transformants were obtained with different plasmids as covalently closed circular molecules and as linearized DNA. The influence of recipient strain and plasmid type as well as of cell number and parameters of the supplied electrical pulse on the transformation efficiency have been investigated. A maximum of 7000 transformants per 100ng of plasmid DNA was reached. The efficiency of transformation was compared with that of the LiCl method.
Disruption and mapping of IDI1, the gene for isopentenyl diphosphate isomerase in Saccharomyces cerevisiae
Authors: Mayer Matthias P. , Hahn Frederick M. , Stillman David J. , Poulter C. Dale
Published: May 2005

Isopontenyl diphosphate isomerase catalyses an essential activation step in the isoprene biosynthetic pathwasy. The Saccharomyces cerevisiae gene for isomerase, IDI1, was recently isolated and characterized (Anderson et al. J. Biol. Chem. 1989aa, 264, 19169–19175). Wild‐type IDI1 was disrupted with a LEU2 marker, and the resulting DNA was used to transform a yeast leucine auxotroph. Southern blots of EcoRI fragments of chromosomal DNA from the diploid strain showed the expected fragments for intact and disrupted IDI1. Dissection and analysis of tetrads demonstrated that IDI1 is an essential single‐copy gene. A CHEF gel and clone grid filter analysis, followed by chromosomal mapping indicated that the gene is located on chromosome that the gene is located on the chromosome XVI approximately 55 kb contromere proximal to PEP4.
Yeast sequencing reports. Sequence of the genes encoding subunits A and B of the vacuolar H+‐ATPase of Schizosaccharomyces pombe
Authors: Ghislain Michel , Bowman Emma Jean
Published: May 2005

The genes coding subunits A (vma1) and B (vma2) of the vacuolar H+‐ATPase from Schizosaccharomyces pombe were cloned by hybridization to cDNAs of the homologous genes in Neurospora crasa. Both genes are interrupted by introns, two in vma1 and four in vma2. Positions of introns do not appear to be conserved when compared to those of N. crassa. The subunit A gene encodes a single product of 619 amino acids and is not interrupted by the coding sequence for a second product as found for Saccharomyces cerevisiae (Kane, P.K., Yamashiro, C.T., Wolczyk, D.F., Neff, N., Goebl, M., and Stevens, T.H. (1990). Science 250, 651–657).
DIT101 (CSD2, CAL1), a cell cycle‐regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls
Authors: Pammer Manuela , Briza Peter , Ellinger Adolf , Schuster Tillman , Stucka Rolf , Feldmann H. , Breitenbach Michael
Published: May 2005

A mutant screen has been designed to isolate mutants in Saccharomyces cerevisiae deficient in spore wall dityrosine. As shown by electron microscopy, most of the mutant spores lacked only the outermost, dityrosine‐rich layer of the spore wall. Mutant dit101, however, was additionally lacking the chitosan layer of the spore wall. Chemical measurements showed that this mutant does not synthesize chitosan during sporulation. The mutant spores were viable but sensitive to lytic enzymes (glusulase or zymolyase). Unlike most of the dit‐mutants, dit101 did show a distinctive phenotype in vegetative cells: they grew normally but contained very little chitin and were therefore resistant to the toxic chitin‐binding dye, Calcofluor White. The cells showed barely detectable staining of the walls with Calcofluor White or primulin. The decrease in the amount of chitin in vegetative cells and the absence of chitosan in spores suggested that the mutant dit101 could be defective in a chitin synthase. Indeed, a genomic yeast clone harboring the gene, CSD2, sharing significant sequence similarity with yeast chitin synthases I and II (C. E. Bulawa (1992), Mol. Cell. Biol. 12, 1764–1776), complemented our mutant and was shown to correspond to the chromosomal locus of dit101. Thus, the mutations dit101 and csd2 (and probably also call; M. H. Valdivieso et al., (1991), J. Cell Biol. 114, 101–109) were shown to be allelic. The gene was mapped to chromosome II and was located about 3 kb distal of FAL1. Using this DNA clone, a transcript of about 3500–4000 nucleotides was detected. Comparing RNA isolated from vegetative cells and from sporulating cells at different times throughout the sporulation process, no significant differences in DIT101 transcript levels could be detected indicating absence of sporulation‐specific transcriptional regulation. However, the amount of DIT101 transcript changed significantly at different stages of the mitotic cell cycle, peaking after septum formation, but before cytokinesis. As most of the chitin synthesis of vegetative cells occurs at this stage of the cell division cycle, chitin synthesis mediated by DIT101 could be primarily regulated at the level of transcription in vegetatively growing cells.
In Saccharomyces cerevisiae, protein secretion into the growth medium depends on environmental factors
Authors: Rossini Dominique , Porro Danilo , Brambilla Luca , Venturini Marina , Ranzi Bianca Maria , Vanoni Marco , Alberghina Lilia
Published: May 2005

In the budding yeast Saccharomyces cerevisiae the cell wall, mainly composed of mannoproteins and glucans, constitutes a barrier to protein excretion in the growth medium. In this paper we have studied the effects of different environmental parameters on excretion of Escherichia coli β‐galactosidase obtained by exploiting the glucoamylase II signal sequence. Excretion of the unglycosylated β‐galactosidase was detectable only in cells grown in rich medium, was affected by temperature (36°C > 30°C >> 24°C) and slightly stimulated by reducing agents. On the contrary, glycosylated proteins, such as α‐galactosidase and glucoamylase II, were excreted to a good extent under all tested conditions of medium composition, growth temperature and pH. These data indicate that optimization of environmental parameters may help the excretion of heterologous proteins, offering advantages for protein purification.
Galactose inhibition of the constitutive transport of hexoses in Saccharomyces cerevisiae
Authors: Nevado Julian , Navarro Ma Asuncion , Heredia Claudio F.
Published: May 2005

The relationship between the pathways of glucose and galactose utilization in Saccharomyces cerevisiae has been studied. Galactose (which is transported and phosphorylated by inducible systems) is a strong inhibitor of the utilization of glucose, fructose and mannose (which have the same constitutive transport and phosphorylation systems). Conversely, all these three hexoses inhibit the utilization of galactose, though with poor efficiency. These cross‐inhibitions only occur in yeast adapted to galactose or in galactose‐constitutive mutants.

The efficiency of galactose as inhibitor is even greater than the efficiencies of each of the other three hexoses to inhibit the utilization of each other. Phosphorylation is not involved in the inhibition and the transport of sugars is the affected step.

The cross‐inhibitions between galactose and either glucose, fructose or mannose do not implicate utilization of one hexose at the expense of the other, as it occurs in the mutual interactions between the latter three sugars. It seems that, by growing the yeast in galactose, a protein component is synthesized, or alternatively modified, that once bound to either galactose or any one of the other three hexoses (glucose, fructose or mannose), cross‐interacts respectively with the constitutive or the inducible transport systems, impairing their function.
Change in transport activities of vacuoles of the yeast Yarrowia lipolytica during its growth on glucose
Authors: Kulakovskaya T. V. , Matyashova R. N. , Shishkanova N. V. , Finogenova T. V. , Okorokov L. A.
Published: May 2005

Vacuoles were isolated from Yarrowia lipolytica yeast cells taken at various growth phases under carbon or nitrogen limitation. Vacuoles from the cells at the logarithmic growth phase showed a high activity of vacuolar H+‐ATPase (0·9–1·1 U/mg protein) and efficiently generated chemical proton gradient and membrane potential across the tonoplast. Ca2+‐ and citrate transport were found to be maximal at this growth phase. At growth retardation and then in the stationary phase all the parameters studied decreased irrespective of the method of growth limitation. The citrate‐transporting activity of vacuoles completely disappeared at growth retardation, also irrespective of the limitation method and irrespective of whether yeast cells overproduced citrate in the culture medium. The citrate‐transporting system of Y. lipolytica vacuolar membrane is concluded not to be involved in citrate efflux and this efflux is probably performed by the plasmalemma transport system.
Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope
Authors: Andreeva Nadezhda A. , Okorokov Lev A.
Published: May 2005

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM‐314 followed by chromatography of the extract on phosphocellulose and QAE‐Sephadex in the presence of 5 mM‐MgCl2, 0·5 mM‐EDTA and 0·1% Triton X‐100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at −20°C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3–208), had low activity for GTP and did not split pyrophosphate, ATP and p‐nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, Km values were 1·7 × 10−4, 1·5 × 10−5 and 8·8 × 10−7 M respectively. Polyphosphatase was most active and stable at pH 6·0–8·0. The enzyme showed maximal activity at 50°C. The time of half inactivation of polyphosphatase at 40, 45 and 50°C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca2+ or Cu2+, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co2+ > Mg2+ > Mn2+ > Fe2+ > Zn2+. Polyphosphatase was completely inhibited by 1 mM‐ammonium molybdate and 50 μM‐Zn2+ or Cu2+ (in the presence of Mg2+).
Cystathionine γ‐lyase of Saccharomyces cerevisiae: Structural gene and cystathionine γ‐synthase activity
Authors: Ono Bun‐Ichiro , Ishii Nobuya , Naito Kazuhide , Miyoshi Shin‐Ichi , Shinoda Sumio , Yamamoto Sumiyo , Ohmori Shinji
Published: May 2005

Purification of Saccharomyces cerevisiae cystathionine γ‐lyase (γ‐CTLase) was hampered by the presence of a protein migrating very close to it in various types of column chromatography. The enzyme and the contaminant were nevertheless separated by polyacrylamide gel electrophoresis. N‐terminal amino acid sequence analysis indicated that they are coded for by CYS3(CYI1) and MET17(MET25), respectively, leading to the conclusion that CYS3 is the structural gene for γ‐CTLase and that the contaminant is O‐acetylserine/O‐acetylhomoserine sulfhydrylase (OAS/OAH SHLase). Based on these findings, we purified γ‐CTLase by the following strategy: (1) extraction of OAS/OAH SHLase from a CYS3‐disrupted strain; (2) preparation of antiserum against it; (3) identification of a strain devoid of the OAS/OAH SHLase protein using this antiserum; and (4) extraction of γ‐CTLase from this strain. Purified γ‐CTLase had cystathionine γ‐synthase (γ‐CTSase) activity if O‐succinylhomoserine, but not O‐acetylhomoserine, was used as substrate. From this notion we discuss the evolutional relationship between S. cerevisiae γ‐CTLase and Escherichia coli γ‐CTSase.
Genetics of heat‐curability of killer virus of yeast
Authors: Weinstein Lee Ann , Capaldo‐Kimball Florence , Leibowitz Michael J.
Published: May 2005

The cytoplasmically inherited M double‐stranded (ds) RNA genome segment of killer virus of Saccharomyces cerevisiae is heat‐curable in some yeast strains but not in others. Temperature sensitivity is conferred on both M1 and M2 dsRNA satellite virus segments by the L‐A‐HN allele of the killer helper virus genome, but not by the L‐A‐H allele. Both diploidy and mating type heterozygosity of the host cell are also correlated with increased virus curability.
Genetic and physical localization of the acetyl‐coenzyme A synthetase gene ACS1 on chromosome I of Saccharomyces cerevisiae
Authors: Steensma H. Yde , Barth Gerold , De Virgilio Claudio
Published: May 2005

The ACS1 gene, encoding acetyl‐coenzyme A synthetase, was mapped genetically at the left arm of chromosome I between pURA3 and PYK1 at 19 and 28 cM respectively. Comparison with the physical map defined a recombinational ‘hot‐spot’ in this region in addition to the one between CDC24 and PYK1.
Genes required for derepression of an extracellular glucoamylase gene, STA2, in the yeast Saccharomyces
Authors: Kuchin S. V. , Kartasheva N. N. , Benevolensky S. V.
Published: May 2005

A diastatic strain of Saccharomyces cerevisiae producing the STA2‐encoded extracellular glucoamylase (GA) in a pronounced glucose‐repressible fashion was used as a parent for generating mutants with reduced GA activity under normal conditions of derepression. In addition to mutations in STA2, five other recessive mutations were identified which fell into four complementation groups designated haf1 through haf4. RNA blot analysis suggested that the haf mutations confer defects in STA2 transcription. The haf mutants were pleiotropically defective in utilization of alternative carbon sources and resembled the snf (sucrose non‐fermenting) mutants identified previously as unable to derepress the expression of the SUC2 gene encoding invertase. We present evidence strongly suggesting that haf1 = snf2, haf3 = snf1 and haf4 = snf5. By phenotypic criteria, the postulated HAF2 gene (which is none of the SNF genes tested) appears to be similar to SNF2, SNF5 and SNF6, and is possibly a non‐redundant extension of this group of functionally related SNF genes.
Para‐aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctional enzyme
Authors: Edman Jeffrey C. , Goldstein Alan L. , Erbe Jennifer G.
Published: May 2005

The Saccharomyces cerevisiae gene for para‐aminobenzoate (PABA) synthase has been identified based upon its ability to confer sulfonamide resistance when present on a multicopy episomal vector. The 3840 bp DNA sequence fragment reported here contains a 2199 bp open reading frame encoding a 733 amino acid protein with similarity to the two components of PABA synthase described for prokaryotes (Escherichia coli PabA and PabB), suggesting that PABA synthase is bifunctional in yeast. The cloned sequence was confirmed to be PABA synthase by gene disruption. Chromosome gel analysis places the gene for PABA synthase on chromosome XIV.
Molecular characterization of the SEC1 gene of Saccharomyces cerevisiae: Subcellular distribution of a protein required for yeast protein secretion
Authors: Egerton Mark , Zueco Jesus , Boyd Alan
Published: May 2005

Strains of Saccharomyces cerevisiae harbouring temperature‐sensitive mutations in the SEC1 and SEC5 genes exhibit an accumulation of post‐Golgi secretory vesicles at 37°C. We have cloned a fragment of yeast DNA which carries two distinct genes, one of which complements a sec1 mutation, and the other a sec5 mutation. Genetic tests confirm that the sec1‐complementing gene is indeed SEC1, and is essential for cell growth. Nucleotide sequence analysis reveals that the cloned SEC1 gene is the same as a previously sequenced sec1‐complementing gene. The SEC1 sequence encodes a protein of 724 amino acids with a predicted molecular mass of 83 kDa. Antibodies purified from a polyclonal antiserum raised against the protein product of the cloned gene recognize a yeast protein of apparent molecular mass 78 kDa which is found in a detergent‐resistant association with a rapidly sedimenting yeast subcellular fraction, behaviour which is suggestive of an interaction with a component of the yeast cytoskeleton.
Cloning and sequencing of the Saccharomyces cerevisiae gene LYP1 coding for a lysine‐specific permease
Authors: Sychrova H. , Chevallier M. R.
Published: May 2005

The LYP1 gene of Saccharomyces cerevisiae was cloned by complementation in lysine‐permease‐deficint recipient yeast cells, and its nucleotide sequence was determined. An open reading frame of 1833 nucleotides was found encoding a polypeptide of 611 amino acids, with a calculated molecular weight of 68 118. Analysis of the deduced primary structure of the protein revealed ten membrane‐spanning regions and three potential N‐glycosylation sites. Analysis of the deduced sequence of protein LYP1 indicates homology with other yeast amino‐acid permeases, in particular with CAN1, and also the lysine‐specific permease of Escherichia coli. The strain transformed by a multi‐copy plasmid harbouring the LYP1 gene, showed a 20‐fold increase in the maximum velocity of lysine uptake over that in the wild type, with no changes in the affinity of the permease for its substrate.
Flocculation of Kluyveromyces marxianus is induced by a temperature upshift
Authors: Fernandes P. A. , Moradas‐Ferreira P. , Sousa M.
Published: May 2005

An upshift of the growth temperature from 26 to 40°C in the presence of calcium leads to the aggregation of Kluyveromyces marxianus cells and to the formation of flocs. Analysis of cell wall proteins, either by sodium dodecyl sulphate–polyacrylamide gel electrophoresis of extractable mannoproteins or by immunolocalization, revealed an accumulation of a protein with Mr 37 kDa (p37), upon flocculation. Immunological studies confirmed the homology of this protein with the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). When mRNA isolated from cells growing at 40°C was translated in vitro, a 35 kDa newly labelled protein was synthesized and immunoprecipitation assays showed that this protein is recognized by p37‐antiserum, suggesting that the 35 kDa polypeptide might be an unglycosylated precursor form of p37. The results indicated that the presence of this cell wall mannoprotein closely related to GAPDH is dependent on the growth temperature, suggesting its role as adhesin.
Metabolic studies of a fructose‐intolerant yeast by in vivo31P‐nuclear magnetic resonance spectroscopy
Authors: Doyle Timothy C. , Donaldson Iain A. , Spickett Corinne M. , Ratcliffe R. George
Published: May 2005

The metabolic effects of the administration of fructose to a yeast expressing the cDNA for rat liver ketohexokinase have been investigated by 31P‐nuclear magnetic resonance spectroscopy. Cessation of growth suffered by the yeast on exposure to 5 and 25 mM‐fructose was accompanied by a large accumulation of fructose 1‐phosphate at the expense of cytoplasmic orthophosphate and nucleoside triphosphate. Shifts in resonances were consistent with a drop in cytoplasmic pH. Arresting growth with 1 mM‐fructose, however, did not result in these changes, although a large accumulation of fructose 1‐phosphate occurred which may have been supported by the mobilization of polyphosphates.
Identification of two divergently transcribed genes centromere‐proximal to the ARG4 locus on chromosome VIII of Saccharomyces cerevisiae
Authors: Rocco Vincenzo , Daly Michael J. , Matre Vilborg , Lichten Michael , Nicolas Alain
Published: May 2005

We have sequenced a 3296 bp segment of the chromosome VIII adjacent to the 3′ end of the ARG4 gene. This segment contains two divergently oriented open reading frames (YSC83 and YSC84). Northern blot analysis showed the presence of transcripts corresponding to these two open reading frames in vegetative cells. Levels of these transcripts increase five to ten‐fold during sporulation. These two genes are not essential for vegetative growth or sporulation. Analysis of the putative protein products on the SwissProt database revealed that the C‐terminal region of the Ysc84 protein contains a putative SH3 domain.
A comparative analysis of distinctive features of yeast protein sequences
Authors: Sapolsky Ronald J. , Brendel Volker , Karlin Samuel
Published: May 2005

The recently published sequence of yeast chromosome III (YCIII) provides the longest continuous stretch of a eukaryotic DNA molecule sequenced to date (315 kb). The sequence contains 116 distinct AUG‐initiated open reading frames of at least 200 codons in length, more than 50 of which had not been described previously nor bear significant similarity to known proteins. We have analysed the YCIII known and putative‐protein sequences with respect to significant statistical features which might reflect on structural and functional characteristics. The YCIII proteins have striking similarities and differences in their sequence attribute distributions compared to the corresponding distributions for all available yeast sequences and other protein collections. Nine examples of YCIII proteins with distinctive sequence features are discussed in detail.
Accurate initiation of mRNA synthesis in extracts from Schizosaccharomyces pombe, kluyveromyces lactis and Candida glabrata
Authors: Woontner Michael , Jaehning Judith A.
Published: May 2005

We demonstrate the successful adaptation to other yeast species of a protocol previously described for production of transcriptionally active whole cell extracts from Saccharomyces cerevisiae (Woontner and Jaehning, 1990, J. Biol. Chem. 265, 8979–8982). Extracts prepared from Schizosaccharomyces pombe, Kluyveromyces lactis and Candida glabrata were all capable of initiating transcription from a template containing the S. cerevisiae CYC1 TATA box fused to a G‐less cassette. Transcription in all of the extracts was sensitive to inhibition by α‐amanitin, indicating that it was catalysed by RNA polymerase II, and was dramatically stimulated by the chimeric activator GAL4/VP16. The different extracts used different subsets of a group of three initiation sites.
The complete sequence of a 15 820 bp segment of Saccharomyces cerevisiae chromosome XI contains the UBI2 and MPL1 genes and three new open reading frames
Authors: Bou German , Esteban Pedro F. , Baladron Victoriano , Gonzalez Gloria A. , Cantalejo Jesus Garcia , Remacha Miguel , Jimenez Antonio , Del Rey Francisco , Ballesta Juan P. G. , Revuelta José L.
Published: May 2005

As part of the EEC yeast genome program, a fragment of 15 820 bp from the right arm of Saccharomyces cerevisiae chromosome XI has been sequenced. This fragment corresponds roughly to the centromere‐distal half of cosmid pUKG046 and to a small fragment of cosmid pUKG096, which are located approximately 150 kb from the centromere. It contains four open reading frames (ORFs) which encode potential proteins of more than 100 amino acid residues, as well as the UBI2 gene which carries an intron and does not show up as an ORF in the sequence analysis programs. One of the putative proteins, YKR412, is very rich in serine and has significant homology at the carboxyl end to Nopp140 phosphoprotein. YKR413 has several predicted transmembrane domains. YKR15, which has been recently cloned as the MPL1 gene, encodes a polypeptide that shows homologies to myosin heavy chain and to the cytoskeleton protein Uso1.
Inhibition of protein synthesis disrupts the golgi apparatus in the fission yeast, Schizosaccharomyces pombe
Authors: Ayscough Kathryn , Warren Graham
Published: May 2005

Schizosaccharomyces pombe was treated with either cycloheximide or anisomycin at levels sufficient to inhibit >95% of protein synthesis for periods upon to 3 h, equivalent to one cell cycle. Treatment for as little as 1 h caused significant loss of the Golgi apparatus by both immunofluorescence and electron microscopy. The loss was quantitated by stereology on electron micrographs. Nearly 90% of the stacked Golgi was lost over a 3 h period. No other intracellular membrane compartment seemed to be affected. Measurement of enzyme activities confirmed these observations. The activity of a resident of the Golgi apparatus, α‐1,2 galactosyltransferase, was reduced over this time, whereas the endoplasmic reticulum marker, BiP, and the cytoplasmic enzyme, hexokinase, were unaffected. The morphological changes associated with cycloheximide addition were reversed on its removal, though there was a lag before cells recommenced growth or secretion of the enzyme, acid phosphatase.
A physical comparison of chromosome III in six strains of Saccharomyces cerevisiae
Authors: Wicksteed Barton L. , Collins Irene , Dershowitz Ann , Stateva Lubomira I. , Green Rebecca P. , Oliver Stephen G. , Brown Alistair J. P. , Newlon Carol S.
Published: May 2005

We have tested the clones used in the European Yeast Chromosome III Sequencing Programme for possible artefacts that might have been introduced during cloning or passage through Escherichia coli. Southern analysis was performed to compare the BamHI, EcoRI, HindIII and PstI restriction pattern for each clone with that of the corresponding locus on chromosome III in the parental yeast strain. In addition, further enzymes were used to compare the restriction maps of most clones with the map predicted by the nucleotide sequence (Oliver et al., 1992). Only four of 506 6‐bp restriction sites predicted by the sequence were not observed experimentally. No significant cloning artefacts appear to disrupt the published sequence of chromosome III. The restriction patterns of six yeast strains have also been compared. In addition to two previously identified sites of Ty integration on chromosome III (Warmington et al., 1986; Stucka et al., 1989; Newlon et al., 1991), a new polymorphic site involving Ty retrotransposition (the Far Right‐Arm transposition Hot‐Spot, FRAHS) has been identified close to CRY1. On the basis of simple restriction polymorphisms, the strains S288C, AB972 and W303‐1b are closely related, while XJ24‐24a and J178 are more distant relatives of S288C. A polyploid distillery yeast is heterozygous for many polymorphisms, particularly on the right arm of the chromosome.