摘要:

AbstractSchizosaccharomyces pombecontains singlerasoncogene homologue,ras1, that functions in the signal transduction pathway conducting the cell's mating processes. To understand the biochemical basis of yeast ras proteins, we have purified the ras1 protein and compared the major biochemical constants with those of RAS2 protein fromSaccharomyces cerevisiaeand mammalian ras proteins. The purified ras1 protein showed a remarkably highKdvalue for GDP binding (178 nM) and for binding with ATP. In contrast, theKdvalue for GTP binding and the rate of GTPase activity were 64 nM and 77 × 10−6s−1at 37°C, respectively; both were higher than normal p21rasprotein, but at the same level as the RAS2 protein. We directly measured rate of GTP binding and GDP binding which were 3.9 × 10−3s−1and 1.8 × 10−3s−1at 30°C, respectively. On the other hand, exchange rates between bound and free nucleotides remained almost constant throughout the tested combination of GTP and GDP, and were several‐fold lower than the binding rate. These results suggest that the release of the guanine nucleotide is the rate‐limiting step in the ras–GTP/GDP cycle. As a whole, the biochemical properties of the ras1 protein are close to those of the RAS2 protein, although these two proteins function differently in the signal transductio

ISSN:0749-503X    

DOI:10.1002/yea.320110902

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe sequencing of a 6619 bp region encoding for a flocculation gene previously cloned from a strain defined asFLO5(Bidardet al., 1994) has revealed that it was aFLO1gene. TheFLO1gene product has been localized at the cell surface of the yeast cell by immunofluorescent microscopy. The Flo1 protein contains four regions with repeated sequences which account for about 70% of the amino acids of this protein. A functional analysis of the major repeated region has revealed that it plays an important role in determining the flocculation level. A gene disruption experiment has shown that theFLO5strain STX 347‐1D contains at least two flocculation genes of theFLO1type but that they are supposed to be inactive and do not contribute to its flocculation. However, enzyme‐linked immunosorbent assays performed on intact cells have revealed that a protein expressed at the cell surface of theFLO5strain STX 347‐1D is antigenically related to Flo1p. A deletion analysis of the 5′ region of theFLO1gene has shown that the expression is submitted to controls which depend on the genetic background of the

ISSN:0749-503X    

DOI:10.1002/yea.320110903

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractSeventy‐six red adenine mutants ofKluyveromyces lactiswere isolated. By complementation they could be assigned to two groups with 31 and 45 mutants. Transformation of several strains from each group with plasmids containing theSaccharomyces cerevisiae ADE1orADE2gene showed that the largest group wasade2and the other group wasade1. Several previously isolated ‘ade1’ mutants were classified to either group and given new gene and allele numbers.ADE1was localized at chromosome III, closely linked to the mating type gene, making it a convenient marker for mating type.ADE2was localized at chromos

ISSN:0749-503X    

DOI:10.1002/yea.320110904

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractIn order to clarify the relationship between salt‐tolerance ofZygosaccharomyces rouxiiand the function of Na+/H+‐antiporter, a gene was isolated fromZ. rouxiiwhich exhibited homology to the Na+/H+‐antiporter gene (sod2) fromSchizosaccharomyces pombe. This newly isolated gene (Z‐SOD2) encoded a product of 791 amino acids, which was larger than the product encoded by itsSz. pombehomologue. The predicted amino‐acid sequence of Z‐Sod2p was highly homologous to that of theSz. pombeprotein, but included an extra‐hydrophilic stretch in theC‐terminal region. The expression ofZ‐SOD2was constitutive and independent of NaCl‐shock.Z‐SOD2‐disruptants ofZ. rouxiidid not grow in media supplemented with 3M‐NaCl, but grew well in the presence of 50% sorbitol, indicating that the function ofZ‐SOD2was closely related to the salt‐tolerance ofZ. rouxii. Several genes are also compared and discussed in relation to the salt‐tolerance ofZ. rouxii. The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the fol

ISSN:0749-503X    

DOI:10.1002/yea.320110905

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractAn NAD+‐dependent D‐arabinitol dehydrogenase (polyol dehydrogenase) gene was isolated fromPichia stipitisCBS 6054 and cloned inSaccharomyces cerevisiae. The gene was isolated by screening of a λ‐cDNA library with a zymogram technique.D‐Arabinitol, xylitol,D‐glucitol and galactitol are substrates for the recominant protein. WithD‐arabinitol as substrate the reaction product isD‐ribulose. The molecular weight of the native tetramer enzyme is 110 000 Da and the monomer is 30 000 Da. The amino acid sequence is homologous to the short‐chain dehydrogenase family. It is 85·5% identical to aD‐arabinitol dehydrogenase fromCandida albicans. The gene inP. stipitiswas induced byD‐arabinitol andP. stipitiswas able to grow onD‐arabinitol. The physiological role ofD‐rabinito

ISSN:0749-503X    

DOI:10.1002/yea.320110906

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe antifreeze peptide AFP6 from the polar fishPseudopleuronectus americanushas been expressed in and secreted by the yeastSaccharomyces cerevisiaeas a biologically active molecule. The gene for the 37 amino acid long peptide has been chemically synthesized using yeast preferred codons. Subsequently, the gene has been cloned into an episomal expression vector as well as in a multicopy integration vector, which is mitotically more stable. The expression is under the control of the inducibleGAL7promoter. The enzyme α‐galactosidase has been investigated as a carrier protein to facilitate expression and secretion of AFP. In order to reach increased expression levels, tandem repeats of the AFP gene (up to eight copies) have been cloned. In most cases the genes are efficiently expressed and the products secreted. The expression level amounts to approximately 100 mg/l in the culture medium. In a number of genetic constructs the genes are directly linked and expressed as AFP multimers. In other constructs linker regions have been inserted between the AFP gene copies, that allow the peptide to be processed by specific proteinases, either from the endogenous yeast proteolytic system or from a non‐yeast source. The latter requires a separate processing step after yeast cultivation to obtain mature AFP. In all these cases proteolytic processing is incomplete, generating a heterogeneous mixture of mature AFP, carrier and chimeric protein, and/or a mixture of AFP‐oligomers. The antifreeze activity has been demonstrated for such mixtures as well as for AFP mul

ISSN:0749-503X    

DOI:10.1002/yea.320110907

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractIn a coordinated approach, several laboratories sequencedSaccharomyces cerevisiaechromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II betweenRPB5andCDC28. The fragment contains four open reading frames probably encoding proteins of 79·2 kDa (corresponding geneYBR156c), 12·1 kDa (YBR157c), 62·7 kDa (YBR158w) and 38·7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found forYBR157c, encoding the smallest open reading frame investigated. Gene replacement within theYBR156cgene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grr1 (Cat80) protein with respect to the leucine‐rich region. Cells harboring a mutation in theYBR158wgene showed strongly reduced growth as compared to the wild‐type cells. The protein predicted fromYBR159wshared 33% identical amino acid residues with the human estradiol 17‐beta‐hydroxysterol dehydrogenase 3. Haploidybr159cmutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild‐type strains. The DNA sequence was deposited at the EMBL data base with accession numbers Z36025 (YBR156c), Z36026 (YBR157c), Z36027 (YBR158w) and Z36

ISSN:0749-503X    

DOI:10.1002/yea.320110908

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromsome X was determined from an ordered set of subclones. The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons. Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to theSME1gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents theMEF2gene probably encoding a second mitochondrial elongation factor‐like protein, D678 is identical to the yeastGSH1gene encoding γ‐glutamylcysteine synthetase and B746 is identical to theCSD3gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation. The deduced amino acid sequence of A550 is 63% identical to the Ccη subunit of a murine TCP‐1‐containing chaperonin and more than 35% identical to thermophilic factor 55 fromSulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP‐1 family. Open reading frame F551 exhibits homology to two regions of theDAL80gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein. In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI. Finally, the sequence contained a tRNAArg3(AGC) gene. The nucleotide sequence data reported in this paper have been deposited in the EMBL and GenBank databases under the accession nu

ISSN:0749-503X    

DOI:10.1002/yea.320110909

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractA new 1150 amino acids long open reading frame (ORF), coding for an essential protein of unknown function was foundSaccharomyces cerevisiaeby sequencing 3754 bp of geonomic DNA. The clone was isolated in a search for a fatty acid‐binding protein (FABP) and was localized on chromosome IX. The ORF bears no homology to FABP, but it shows weak similarity toPlasmodium vivaxreticulocyte binding protein 1 and to aggregation‐specific adenylate cyclase fromDictyostelium discoideum. The new gene is constitutively transcribed regardless of the carbon source used. The nucleotide sequence reported in this paper has been deposited in GenBank (Accession Number U179

ISSN:0749-503X    

DOI:10.1002/yea.320110910

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

ISSN:0749-503X    

DOI:10.1002/yea.320110911

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY