摘要:

AbstractWe have determined the structure of the allantoin permease (DAL4) gene ofSaccharomyces cerevisiae. The gene patatively encodes a hydrophobic protein with a Mrof 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 byDAL4is its striking similarity to the uracil permease component encoded byFUR4Although 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 ofDAL4contains all lof the characterizedcis‐acting elements previously reported for inducible allantoin pathway genes: six sequences homologous toUASNTR, the element responsible for nitrogen catabolite repression‐sensitive activation of allantoin pathway gene expression, and two sequences homologous to thecis‐acting element responsible for inducere‐responsiveness of the allantoin pathway genes,UIS. The finding of these homologous sequences predicted to exist on the basis ofDAL4's expression characteristics, supports and strengthens the suggestion that these elements mediate the functions we have we have previously ascribe

ISSN:0749-503X    

DOI:10.1002/yea.320081202

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY

摘要:

AbstractAn efficient delivery method for introducingin vitrosynthesized RNA into yeast into has been developed using electroporation. Spheroplast preparation, electroporation, and subsequent expression analysis can be accomplished within a single day. The use of introduced mRNA constructs avoids any complications due to nuclear regulation and is particularly suited for cytoplasmic regulatory studies. Moreover, this technique is useful for introduicing those RNas that connot be madein vivo, Such as poly (A)−mRNAs or RNAs with base modifications. We demonstrate that theEscherichia coli GUSgene and the fireflyLucgene are both excellent reporter genes for RNA electroporatio

ISSN:0749-503X    

DOI:10.1002/yea.320081203

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY

摘要:

AbstractSterol auxotrophic strains ofSaccharomyces cerevisiaewere grown and allowed to conjugate on media supplemented with various sterols.The mating efficiency of the auxotrophs is perturbed by the relacement of the normal yeast sterol, ergsterol, with other sterols. After 4 h of mating, cells grown on ergosterol a 30‐fold higher productive mating efficiency than those cells grown in stigmasterol. Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non‐ergosterol sterols. Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol‐grown cells to undergo cytoplasmic fusion during conjugation. Many of the mated pairs remained adherent but Prezygotic even after 12 h of incubation. The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal bu

ISSN:0749-503X    

DOI:10.1002/yea.320081204

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY

摘要:

AbstractThe transport system for malic acid present inSchizosaccharomyces pombecells, growing in batch culture on several corbon sources, has been studied. It was found that the diarboxylic acid crrier ofS. Pombeis a proton‐dicarboxylate symporter that allows transport and accumulation as a function of ΔpH with the following kenetic parameters at pH 5·0:Vmax= 0·01 nmol of total malic acids−1mg (dry weight) of cells,−1andKm= 0·1mMtotal malica acid uptake (pH 5·0) was accompanied by desappearance of extracellular protons, the uptake rates of which followed Michaelis‐Menten kinetics as a function of the acid conscentration. TheKmvalues, calculated as the concentrations either of anions or of undissociated acid, at various extracellular pH values, pointed to the monoanionic form as the transported species. Furthermore, accumulated free acid suffered rapid efflux after the addition of the portonophore carbonyl cyanidm‐chlorophenyl hydrazone. These results suggested that the transport system was a dicarboxylateproton symporter. Growth of cells in a medium wiht glucose (up to 14%, w/v) and malic acid (1·5%, w/v) also resulted in proton‐dicarboxylate activity, suggesting that the system, besides being constitutive, was still active at high glucose concentratons. The following dicarboxylic acids acted as competitive inhibitors of malic acid transport at pH 5·0:D‐ malic acid, succinic acid, fumaria acid oxaloacetic acid, α‐Ketoglutaric acid, maleic acid, maleic and malonic acid. In addition all of these dicarboxylic acids induced proton movements that followed Michaelis–Menten kinetics. It was concluded that the malic negatively charged form (probably the monoanionic form) was transported by a proton‐symport mechanism and that the carrier appeared to be a common ‘dicarboxylat transport sysmem’. The undissociated acid entered the c

ISSN:0749-503X    

DOI:10.1002/yea.320081205

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY

摘要:

AbstractCells of Saccharomyces cerevisiae 179‐5, an ornithine decarboxylase mutant (spe‐1), showed several ultrastructural abnormalities when cultivated in the absence of polyamines. Besides the appearance of microvacuole‐like spaces in the cytoplasm and of deformed nuclei, the most important alterations seemed to be located in the cell wall, which was thicker and of heterogeneous texture, and in the cell membrane, of irregular contour. These modifications could not be evoked by general stress conditions elicited by lack of nutrients. The relative levels of cell wall polysaccharides were altered in polyamine‐deprived organisms, giving an envelope with increased mannan and decreased glucan content; this cell wall was incompletely attacked by the lytic enzyme zymolyase. Polyamine depletion led also to some abnormalities in the budding pattern. The above observations suggest the involvement of polyamines in the correct structure and organization of the yea

ISSN:0749-503X    

DOI:10.1002/yea.320081206

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY

摘要:

AbstractA DNA fragment ofSaccharomyces cerevisiaewith high homology to the acetyl‐coenzyme A (acetyl‐CoA) synthetase genes ofAspergillus nidulansandNeurospora crassahas been cloned, sequenced and mapped to chromsome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79·2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons dowunstream, is in a more conventional context. Possible implications of two alternative traslational start sites for the celular lacalization of the enzyme are disussed. A stable mutant of this gene, obtained by the gene disruptiona technique, had the same low basal acitivity of acetyl‐CoA synthetase as wild‐type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetlyl‐CoA synthetase (ACSI) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction os isocitrate lyase on acetate media, indicating that activity of acetyl‐CoA sytheatase is dispensable for incuctionof the glyoxylate cycle inS. cerevisiae. Surprisingly, disruption of theACSIgene did not affect growth on media containing ethanol as the sole crbon source. Demonstrating that threre are alternative pathways pathways leading to acetyl‐CoA under the

ISSN:0749-503X    

DOI:10.1002/yea.320081207

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY

摘要:

AbstractAt‐pH 5–6 ATP‐depleted washed cell preparations of strain NC233‐10b[pII4‐9], in which the cytosine permease was overexpressed, absorbed cytosine, hypoxanthine or fluorocytosine stoichiometrically with, respectively, about 1, 1·4 and 5 proton equivalents. The cellular pH fell proportionately. The membrane depolarization caused by each compound was assayed in the presence of glucose with a voltage‐sensitive dye and increased in the same order. Fluorocytosine significantly lowered the growth yield that a ‘petite’ strain of the yeast formed at limiting glucose concentrations. At pH 5·6 with extracellular [K+] below 1 mM, each of the three substrates was accumulated about 200‐fold from a dilute solution at the expense of the proton gradient. This concentration ratio corresponds to a solute gradient (Δμs) of 13 kJ mol−1. Raising [K+]0systematically lowered the substrate accumulation ratio and ΔμH. The mean ratio Δμs/ΔμHwas 0·82 all three substrates. It was concluded that whereas the behaviour of cytosine approximated to that expected for a symport of unit proton stoichiometry, the absorption of protons with fluorocytosine and, to a lesser extent, hypoxanthine, was only partly conserved as useful work. A possible mechanism of thi

ISSN:0749-503X    

DOI:10.1002/yea.320081208

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY

摘要:

AbstractThe alkane‐assimilating yeastCandida tropicaliswas used as a host for DNA transformations. A stableade2mutant (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 ofC. albicansand caries anADE2gene marker and aCanadidaautonomously 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 inEscherichia coli. A second vector, pADE2, containing the isolatedC. tropicalis ADE2, gene was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of theade2locus. Different integration types were observed at one or bothade2alleles in single or in tandem re

ISSN:0749-503X    

DOI:10.1002/yea.320081209

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY

摘要:

AbstractIn this study, glucose repression inSaccharomyces cerevisiaewas analysed under defined physiological conditions, at both the molecular and physiological levels, by pulsing glucose to a galactose‐limited continuous culture. During this pulse of glucose, the galactose feed was kept constant. Directly after the glucose pulse, carbon dioxide production increased while oxygen consumption remained constant, demonstrating that the surplus of glucose had been consumed by means of fermentation. The direct accumulation of galactose in the medium after the glucose pulse indicated that the consumption of galactose had been stopped instantaneously. Galactose uptake experiments revealed that the galactose transporter was still present but apparently was incapable of galactose uptake, which could be due to inhibition of the galactose transporter by glucose. The total concentration of cAMP increased from 5 nmol g−1att= 0 to 25 nmolg−1att= 1·5 min. After 2 min the concentration of cAMP gradually decreased again to the normal level. Within 2 min after the addition of glucose, the transcription of theGALgenes andSUC2was inhibited. In addition, the transcription of theHXK1gene, encoding hexokinase isoenzyme 1, was also inhibited, which demonstrates that theHXK1gene is regulated at the transcriptional level comparable with inv

ISSN:0749-503X    

DOI:10.1002/yea.320081210

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY

摘要:

AbstractA mutant screen has been designed to isolate mutants inSaccharomyces cerevisiaedeficient 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. Mutantdit101, 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,dit101did 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 mutantdit101could 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 ofdit101. Thus, the mutationsdit101andcsd2(and probably also call; M. H. Valdiviesoet 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 ofFAL1. 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 inDIT101transcript levels could be detected indicating absence of sporulation‐specific transcriptional regulation. However, the amount ofDIT101transcript 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 byDIT101could be primarily regulated at the level of transcription in vegetatively

ISSN:0749-503X    

DOI:10.1002/yea.320081211

出版商:John Wiley&Sons, Ltd.    

年代:1992

数据来源: WILEY