摘要:

AbstractWe have analyzed by genetic means 43 strains of Saccharomyces that had been isolated from fermenting grape musts in Italy. Twenty eight of these strains were isolated from 28 cellars in the Region of Emilia Romagna. The other 15 strains came from 5 fermentations at four cellars near the city of Arpino, which is located south and east of Rome.We found that 20 of the 28 strains from Emilia Romagna were heterozygous at from one to seven loci. The balance were, within the limits of our detection, completely homozygous. All these strains appeared to be diploid and most were homozygous for the homothallism gene (HO/HO). Spore viability varied greatly between the different strains and showed an inverse relation with the degree of heterozygosity.Several of the strains, and in particular those from Arpino, yielded asci that came from genetically different cells. These different cells could be interpreted to have arisen from a heterozygote that had sporulated and, because of the HO gene, yielded homozygous diploid spore clones. We propose that natural wine yeast strains can undergo such changes and thereby change a multiple heterozygote into completely homozygous diploids, some of which may replace the original heterozygous diploid. We call this process ‘genome renewal

ISSN:0749-503X    

DOI:10.1002/yea.320101203

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractIncubation of starved galactose‐grownS. cerevisiaecells with cyanide reduced glucose uptake as measured over a 5‐s period. TheVmaxfor glucose uptake was decreased by over a factor of two but the apparent affinity for glucose doubled. When measured in the sub‐second time scale, however, there was no significant inhibition of glucose uptake, by cyanide, up to 200‐ms, clearly demonstrating that, in cyanide treated cells, glucose uptake was not linear for the first 5‐s.After a 200‐ms exposure of untreated cells to radio‐labelled glucose, less than 10% of the intracellular label resided in soluble uncharged compounds. In cyanide‐treated cells up to 43% of the labelled compounds were uncharged, with a concurrent reduction of intracellular label residing in anionic compounds. The results suggest that, in the presence of 10 mM cyanide when respiration is inhibited, a reduction in the cellular ATP concentration causes a reduction in hexose‐kinase activity which results in an accumulation of internal free glucose, which in turn causes a reduction in net

ISSN:0749-503X    

DOI:10.1002/yea.320101204

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractAnalysis of the DNA sequences of new members of theSaccharomyces cerevisiae MEL1‐MEL10gene family showed high homology between the members. TheMELgene family, α‐galactosidase‐coding sequences, have diverged into two groups; one consisting ofMEL1andMEL2and the other ofMEL3‐MEL10. In twoS. cerevisiaestrains containing five or sevenMELgenes each, all the genes are nearly identical, suggesting very rapid distribution of the gene to separate chromosomes. The sequence homology and the abrupt change to sequence heterogeneity at the centromere‐proximal 3′ end of theMELgenes suggest that the distribution of the genes to new chromosomal locations has occurred partly by reciprocal recombination at solo delta sequences.We identified a new open reading frame sufficient to code for a 554 amino acid long protein of unknown function. The new open reading frame (Accession number Z37509) is located in the 3′ non‐coding region ofMEL3‐MEL10genes in opposite orientation to theMELgenes (Accession numbers Z37508, Z37510, Z37511). Northern analysis of total RNA showed no hybridization to a homologous probe, suggesting that the gene is not expressed eff

ISSN:0749-503X    

DOI:10.1002/yea.320101205

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractRecombinant human haemoglobin A (rHbA) was produced by a leucine‐requiring strain ofHansenula polymorphawhich had been transformed with an integration vector containing theSaccharomyces cerevisiae LEU2gene and cDNAs for the expression of α and β globin each driven by theH. polymorpha MOXpromoter. After 40 generations in a chemostat it was found that the integrated vector had become amplified in the host strain. In some cases this led to an increase inLEU2gene dosage, but a loss of globin expression cassettes. In other cases the globin gene dosage also increased. These changes coincided with an increase in rHbA production in the culture, which was reversed when the dilution rate was increased. Isolates from a chemostat culture producing elevated levels of rHbA were grown in fed‐batch fermentations, resulting in higher productivities than when inoculated with the parent strain. The rHbA produced was purified and characterized. Oxygen binding studies and electrospray mass spectrometry showed that the rHbA had been processed and assembled correctly, and behaved as a fully functional co‐operative t

ISSN:0749-503X    

DOI:10.1002/yea.320101206

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have isolated a pyruvate decarboxylase (PDC) gene from the yeastHanseniaspora uvarumusing theSaccharomyces cerevisiae PDC1gene as a probe. The nucleotide sequence of this gene was determined and compared toPDCgenes from yeast and other organisms. TheH. uvarum PDCgene is more than 70% identical to theS. cerevisiaePDC isozymes and possesses a putative thiamine diphosphate binding site. The PDC enzyme was purified and partially characterized. TheH. uvarumPDC was very similar to other known PDCs; theKmfor pyruvate was 0·75 mM, and the enzyme is a homotetramer with subunits ofMr= 57 000. The sequence has been submitted to GenBank under Accession No. U13635

ISSN:0749-503X    

DOI:10.1002/yea.320101207

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractPulse‐chase experiments with [14C]glucose demonstrated that in the cell wall of wild‐typeSaccharomyces cerevisiaealkali‐soluble (1–3)‐β‐glucan serves as a precursor for alkali‐insoluble (1–3)‐β‐glucan. The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali‐insoluble glucan. These cells, unable to form a glucan–chitin complex, were highly vulnerable to hypo‐osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall.Conversion of alkali‐soluble glucan into alkali‐insoluble glucan occurred both early and late during budding and also in the ts‐mutantcdc24

ISSN:0749-503X    

DOI:10.1002/yea.320101208

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThis communication describes the development of genetic tools for the yeastYamadazyma ohmeri. Nystatin enrichment proved highly effective for isolating various auxotrophic strains, which were classified by complementation analysis. Biosynthetic genes encoding known biochemical functions were isolated by polymerase chain reaction, includingYoLEU2andYoURA3that were sequenced. Using these homologous genes as selective markers, DNA transformation was accomplished by electroporation. Transformation with pBR322‐based plasmids, cut within the coding region of the homologous marker gene, yielded 20 to 50 stable transformants per μg of DNA. In about 80% of the cases, integration of plasmid DNA sequence occurred by homologous recombination of a single plasmid into the chromosome. Excision of the plasmid permitted gene replacement, as illustrated by the substitution of a wild‐typeURA3gene by anin vitrogenerated deletion.Sequences conferring extrachromosomal replication were isolated fromY. ohmeriDNA. Plasmids based on pBR322 carrying such anARSand either selective markers transformed at 104/μg and were shown to replicate freely inY. ohmeriat an approximate copy number of 40. Unexpectedly, we observed that BS‐SKRderivatives carrying eitherYoLEU2orYoURA3but noY. ohmeri ARSalso replicated extrachromosomally. Linearization of transforming plasmids within regions homologous or not to chromosomal sequences stimulated transformation frequencies up to four‐fold. The sequences are available for consultation under EMBL accession number Z35101 forYoLEU2and Z35100

ISSN:0749-503X    

DOI:10.1002/yea.320101209

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe Pas1 protein (Pas1p) is required for peroxisome biogenesis inSaccharomyces cerevisiaeand contains two putative ATP‐binding sites, each within a domain which is conserved among members of the recently characterized AAA‐family. To elucidate whether both putative ATP‐binding sites are essential for Pas1p function, lysine467of the first and lysine744of the second putative ATP‐binding site were each changed to glutamate by site‐directed mutagenesis. While replacement of lysine744abolished the function of the Pas1 protein in peroxisome biogenesis, replacement of lysine467had no obvio

ISSN:0749-503X    

DOI:10.1002/yea.320101210

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractTwo fragments containing both an autonomous replicating sequence (ARS) and a centromere have been isolated and sequenced from the yeastKluyveromyces marxianus. The ARS and centromeric core sequences are only 500 bp apart, but ARS activity could be separated from the centromeric sequences.Centromeric sequences are organized in a similar way to those of budding yeasts: two well‐conserved elements: CDEI (5′ TCACGTG 3′) and CDEIII (5′ TNTTCCGAAAGTWAAA 3′), are separated by a 165 bp AT‐rich (± 90%) CDEII element whose length is twice that ofSaccharomyces cerevisiaeCDEII but almost identical to that ofK. lactis.The ARS‐core consensus sequence (5′ TTTATTGTT 3′) is also similar to that ofK. lactis. Both ARS and centromeric elements function in this strain, albeit inefficiently, but not inS. cerevisiae.A third ARS‐containing fragment with a different organization has been isolated and sequenced.The nucleotide sequences of DNA fragments reported in this paper will appear in the EMB data library under the accession numbers: Z3

ISSN:0749-503X    

DOI:10.1002/yea.320101211

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractCharacterization of cdk (cyclindependentkinases) substrates and studies of their regulation require purified enzymatic complexes of cdc2‐related catalytic and cyclin regulatory subunits. We produced human Cdc2 kinase in the fission yeastSchizosaccharomyces pombeas a fusion protein with glutathione S‐transferase (GST). The GST‐human Cdc2p fusion protein was activein vivosince it rescued a temperature‐sensitive allele of cdc2. The fusion protein was purified using a one‐step chromatography procedure with glutathione–Sepharose and exhibited a catalytic activityin vitro. Yeast cyclin B and suc1 were found in association with GST‐Cdc2. A 17‐fold stimulation of GST‐Cdc2 kinase activity was obtained by incubation of recombinant human cyclin A with theS. pombecellular extract prior to affinity purification. This indicates that cyclin concentration is limiting in this overexpression system. These findings describe a fast and easy production of active recombinant human Cdc2 kinase in yeast that can be used for bi

ISSN:0749-503X    

DOI:10.1002/yea.320101212

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY