摘要:

AbstractThenefgene of human immunodeficiency virus type 1 (HIV‐1) has been expressed in the yeastSaccharomyces cerevisiaeto produce native Nef proteins. The proteins of Mr27 kDa and 25 kDa, produced by translation from the first and second start codons of thenefgene react with human HIV‐1 antisera. Under low‐level steady‐state expression conditions, Nef27 undergoes myristylation and is targeted to the nuclear fraction while Nef25 is not myristylated and not nuclear localized. When produced rapidly and to high levels, Nef27 is initially present in the cytoplasm as a soluble myristylated protein that later fractionates with the

ISSN:0749-503X    

DOI:10.1002/yea.320090602

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractTransformedSaccharomyces cerevisiaecells overexpressing theEscherichia coli LacZgene and the transcriptional activatorGAL4, release in the external medium a fraction (from 2 to 10%) of the total β‐galactosidase activity (Porroet 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 activatorGAL4.In the present paper we have characterized theGAL4‐induced cell lysis phenomenon. The expression of theGAL4gene causes morphological modifications and alteration of the cell size distribution. The cell lysis is independent of the expression of the heterologousLacZgene 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 popula

ISSN:0749-503X    

DOI:10.1002/yea.320090603

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have previously shown that the heat‐induced enhanced decay of yeast mRNAs encoding ribosomal proteins (rp‐mRNAs) requires ongoing transcription during the heat treatment [Herrueret 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 thetrans‐acting factor required for the increase in the rate of degradation of rp‐mRNAs upon stress is not on

ISSN:0749-503X    

DOI:10.1002/yea.320090604

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractKluyveromyces lactiskiller toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations inSaccharomyces cerevisiaethat leads to toxin resistance and a temperature‐sensitive phenotype. These mutant cells arrest growth at 37°C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to beCAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23°C and 37°C. Given that theCAL1activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer t

ISSN:0749-503X    

DOI:10.1002/yea.320090605

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractA system is described for the selection of DNA sequences showing promoter activity in the yeastSaccharomyces cerevisiaeusing a heterologous reporter enzyme which is efficiently secreted by the yeast host. A multicopy shuttle plasmid of the YEp‐type was constructed so as to carry multiple unique cloning sites at the 5′ end of theAspergillus awamoriglucoamylase cDNA. Glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a DNA fragment from any source, provided it is endowed with promoter function inS. cerevisiae.As the glucoamylase signal‐peptide is functional inS. cerevisiae, the enzyme is efficiently secreted by the yeast transformants. This phenotype can be very easily detected on plate assays and accurately quantified by spectrophotometric analysis of the culture supernatant. SinceS. cerevisiaenaturally lacks amylolytic activity, any wild‐type strain can be used as a host in this system. To evaluate the system, a DNA pool of random fusions was created by ligatingsau3A digestedS. cerevisiaegenomic DNA to theBglII‐linearized vector. The resulting hybrid plasmids were transformed intoS. cerevisiaeand several transformants secreting glucoamylase to varying degrees were

ISSN:0749-503X    

DOI:10.1002/yea.320090606

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractAn enzymatic end‐point assay of trehalose using acid trehalase from yeast is described. After quantitative hydrolysis of trehalose by acid trehalase, the resulting glucose is assayed with the commercially available glucose oxidase/peroxidase dye system. Pre‐existing glucose is determined in a control reaction from which acid trehalase is omitted. When intact cells are analysed for trehalose, pre‐existing glucose can be washed out with ice‐cold water without reducing the trehalose content of the cells. A convenient method for extraction of trehalose from intact yeast cells is heating for 20 min at 95°C followed by centrifugation. The specificity of the assay is determined by the specificity of the acid trehalase preparation used. As described previously (Mittenbühler, K. and Holzer, H., 1988,J. Biol. Chem.263, 8537–8543; Mittenbühler, K., 1988,Thesis, University of Freiburg), the following sugars and sugar derivatives do not form glucose when incubated with purified acid trehalase: sucrose, cellobiose, mellobiose, raffinose, maltose, lactose, glucose‐6‐phosphate, glucose‐1‐phosphate, galactose. The application of the new trehalose assay to yeast cells grown to different growth stages and at various tempe

ISSN:0749-503X    

DOI:10.1002/yea.320090607

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractHEM6 (HEM12)inSaccharomyces cerevisiaeencodes uroporphyrinogen decarboxylase, the fifth enzyme in the heme biosynthetic pathway. TheHEM6 (HEM12)gene was cloned by complementation of heme auxotrophy of ahem6mutant. Sequence analysis revealed an open reading frame of 1086 nucleotides. The predicted amino acid sequence ofHEM6 (HEM12)shows extensive homology to those reported for uroporphyrinogen decarboxylase from mammalian sources. Expression ofHEM6 (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

ISSN:0749-503X    

DOI:10.1002/yea.320090608

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractThe glucose oxidase gene (god) fromAspergillus nigerwas expressed inHansenula polymorphausing the methanol oxidase promoter and transcription termination region and the MF‐α leader sequence fromSaccharomyces cerevisiaeto direct secretion. The expression cassette was cloned into theS. cerevisiaevector YEp13 and used to transformH. polymorphastrain A16. In the initial transformants plasmid replication was unstable, but was stabilized by a growth regime consisting of alternating cycles of selective and non‐selective growth. The stabilized strain was grown to high cell density by fed‐batch fermentation. Upon induction of theMOXpromoter, glucose oxidase synthesis was initiated. At the end of the fermentation, the culture density was 76 g dry weight/1 and 108 IU/ml (0·5 g/l or 0·65% dry weight) glucose oxidase was found in the culture medium; a further 86 IU/ml (0·43 g/l or 0·56% dry weight) was recovered from the cell lysate. A plate assay was used to monitor glucose oxidase levels in individual colonies. This was then used to isolate mutants which showed abnormal regulation ofgodexpression or which showed an altered pattern of secretion. One mutant, which showed increased production of glucose oxidase, was grown to high cell density by fed‐batch fermentation (100·6 g/l) and produced 445 IU/ml (2·25 g/l or 2·2% dry weight) extracellularly and 76 IU/ml (0·38 g/l or 0·4% dry weight) intracellularly. The mutant thus not only increased total production but exported 83% of the total enzyme made compared to 55% in

ISSN:0749-503X    

DOI:10.1002/yea.320090609

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractAminopeptidase yspI was purified to apparent homogeneity from the fission yeastSchizosaccharomyces pombe. The molecular mass of the native enzyme was estimated to be 184 kDa by gel filtration chromatography. A value of 92 kDa was calculated after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme is thus a dimer with two identical subunits. Optimum pH for cleavage of synthetic aminoacyl‐4‐nitroanilides is 7·0. Mercury ions, EDTA and chloroquine were found to be potent inhibitors of aminopeptidase yspI activity. Substrate specificity studies indicate that the purified enzyme cleaves L‐lysine‐4‐nitroanilide with hig

ISSN:0749-503X    

DOI:10.1002/yea.320090610

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY

摘要:

AbstractWe report the DNA sequence of an 8 kb segment localized on the right arm of chromosome II fromSaccharomyces cerevisiae. The sequence reveals the presence of eight open reading frames (ORFs). Three of them, YBR1402, YBR1405 and YBR1406 are previously sequenced genes, respectively theRIM2(replication in mitochondria),MSI1(multicopy suppressor ofIRA1gene) andPGI1(phosphoglucoisomerase) genes. The predicted product of the ORF YBR1401 could be the putative yeast ribosomal protein L21. A new essential gene, YBR1403, has been identified by disruption; it possesses a leucine zipper motif.

ISSN:0749-503X    

DOI:10.1002/yea.320090611

出版商:John Wiley&Sons, Ltd.    

年代:1993

数据来源: WILEY