摘要:

AbstractThis report describes two sets of plasmid vectors that facilitate the identification of regions of complementation in cloned genomic inserts via transposon or insertional mutagenesis. The first set containsARS‐H4CEN6, a yeast selectable nutritional marker (HIS3, TRP1orURA3), andneofor selection inEscherichia coli. These plasmids lack theTn3transposition immunity region present in pBR322 derived vectors, and are permissive recipients forTn3transposon mutagenesis. The second family of plasmids described facilitate gene disruption procedures performedin vitro. These vectors carry disruption cassettes that contain different yeast selectable markers (HIS3, LEU2, TRP1orURA3) adjacent to theTn5 neogene. These genes can be excised as a cassette on a common restriction fragment and introduced into any desired restriction site with selection for kanamycin resistanc

ISSN:0749-503X    

DOI:10.1002/yea.320101003

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have developed a screening method to isolate yeast genes regulated by a specific transcription activator. The screen is based on the use of expression libraries in which thelacZreporter gene is placed under control of yeast regulatory elements. Two partially representative libraries, constructed by different methods, were used to isolate genes regulated by the yeast CCAAT‐box binding protein Hap2p. Among 26 fusions shown to be regulated by Hap2p onlyCYT1was known to be regulated by this activator. Sequence analysis revealed that most of the remaining regulated fusions are in new yeast genes, while some are in previously characterized yeast genes (PTP1, RPM2, SDH1). Optimal expression of these three genes also requires Hap3p and Hap4p and is regulated by carbon source. Hap2p was known to regulate expression of genes involved in Krebs cycle, electron transport and heme biosynthesis. Our results suggest that Hap2p could play a more general role by regulating other mitochondrial processes such as protein import and phosphate transport (PTP1) or maturation of mitochondrial tRNAs (RPM2). Among the remaining regulated fusions, two of them correspond to open reading frames (ORFs) on chromosomes III and XI whose nucleotide sequences have been entirely determined. The use of this approach to functionally analyse ORFs of unknown function is discusse

ISSN:0749-503X    

DOI:10.1002/yea.320101004

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have studied the phenomenon of infertility of yeast hybrids obtained with physiological conditions under the control of compatible mating systems. The yeasts investigated are threeSaccharomycesspecies:S. cerevisiae, S. uvarumand a new species,S. douglasii. The diploid hybrids from crosses between these species sporulate well but are essentially infertile. The rare viable spores, one per 104to 105asci, that have been examined carry a complete genome comprised of chromosomes contributed by both parents but invariably have extra chromosomes, i.e. they are generally disomic for at least two or three chromosomes. This observation is consistent with a failure, in meiosis I, of the pairing and disjunction of homologous chromosomes which in most cases results in spores with an incomplete set of chromosomes. This apparent lack of pairing of ‘homeologous’ chromosomes in meiosis I was analysed in most detail withS. cerevisiae/S. douglasiihybrids. As a genetic tool we studied frequencies of recombination, taking advantage of anS. douglasiibreeding stock of some 50 identified mutations in non‐switching haploids. Recombination, although markedly reduced, could be observed at both the chromosomal and allelic levels, implying a sporadic pairing in meiosis to allow genetic exchange. Meiotic recombination frequencies were studied for 14 gene pairs and generally found to be reduced ten‐fold. Heteroallelic recombination (gene conversion) frequencies were measured at 22 loci and were judged to be reduced at least two‐ to 100‐fold. DNA hybridization experiments withS. cerevisiaegene probes gave results consistent with low DNA sequence homologies betweenS. cerevisiaeandS. douglasii. Moreover, by chance, our experiments disclosed anotherSaccharomycesstrain (CBS2908, originally classified asS. cerevisiae) with hybridization patterns identical toS. douglasiiexcept for the hybridization with the Ty transposon probes. Crosses between CBS2908 andS. douglasiiyielded diploid hybrids with 80–90% spore viability, thus establishing a second member of theS. dou

ISSN:0749-503X    

DOI:10.1002/yea.320101005

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractProduction of recombinant human serum albumin (rHSA) controlled by the constitutive promoter phosphoglycerate kinase was studied inKluyveromyces lactis. It was governed by both cell concentration and glycolytic flow. The triggering of the fermentation metabolism by unfavourable culture conditions (pH, pO2,D) caused a decrease in the synthesis of the heterologous protein. The highest productivity (75 mg 1−1per h) and rHSA concentration (62 mg 1−1) were obtained in chemostat culture with a dilution rate of 0·12 h−1and with 38 g 1−1dr

ISSN:0749-503X    

DOI:10.1002/yea.320101006

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have developed and evaluated an easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate (SDS)–gel electrophoresis and Western blotting. The procedure comprises a centrifugation step to harvest the cells, addition of a sample buffer and heating, then another centrifugation step before applying the extracted proteins found in the supernatant to an SDS gel. It is applicable to the study of large numbers of samples in 1 day. This procedure is easier, quicker, and as efficient as procedures using base and 2‐mercaptoethanol, but somewhat less efficient than lysis with glass beads under certain conditi

ISSN:0749-503X    

DOI:10.1002/yea.320101007

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe catalytic capacity of several excreted pectinolytic enzymes obtained from various yeast strains was examined usingin vivoand biochemical techniques. Of the 33 yeast strains studied, 30 were isolated from champagne wine during alcoholic fermentation. Only one yeast strain was found to excrete pectinolytic enzymes and was identified asSaccharomyces cerevisiaeand designated SCPP. Pulsed‐field gel electrophoresis and the polymerase chain reaction technique were used to characterize further this specific strain. Three types of pectinolytic enzymes were found to be excreted by SCPP: polygalacturonase, pectin‐lyase and pectin‐esterase. These enzymes allow pectin hydrolysis during cell g

ISSN:0749-503X    

DOI:10.1002/yea.320101008

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractTheLUCgene coding forPhotinus pyralisfirefly luciferase was cloned in different yeast episomal plasmids in order to assess its possibilities as anin vivoreporter gene. Activity of the enzyme in transformed cellsin vivowas measured by following light emission and assay conditions optimized in intact cells, with regard to oxygen concentration, temperature, cell concentration in assay mixtures and external ATP concentration. Among the factors tested, light emission was drastically influenced by the external pH in the assay (which resulted in a ten‐fold amplification signal) and by substrate permeability. The growth phase of the cells was also important for the level of activity detected. Cloning of firefly luciferase gene under the control of different yeast‐regulated promoters (ADH1, GAL1–10) enabled us to measure their strength which correlated well with previously described data. We conclude that firefly luciferase is an adequate gene reporter for thein vivosensitive determination of gene expression and promoter strength in

ISSN:0749-503X    

DOI:10.1002/yea.320101009

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractInSaccharomyces cerevisiae, expression of a gene adjacent to the retrotransposon Ty1 is often mediated by Ty‐internal sequences. We have identified novel mutants,tye7, which are affected in Ty1‐mediated expression ofADH2through a Ty1 sequence distal to the 5′ long terminal repeat sequence. TheTYE7gene has been isolated and characterized. It encodes a 33 kDa protein whose N‐terminal third is extremely rich in serine residues (28%). Within its C‐terminal sequence, a remarkable similarity to Myc and Max proteins can be found. Thus, TYE7 is a potential member of the basic region/helix‐loop‐helix/leucine‐zipper protein family.TYE7function is not essential for growth. It may primarily function as a transcriptional activator in Ty1‐mediated gene expression, as has been confirmed by the activation of reporter gene expression by a LexA‐TYE7 hybrid protein.ADH2activation by defined Ty1 derivatives revealed that TYE7 acts positively through the more distal Ty1 enhancer element (region D), and negatively in a region between A (the 5′ proximal en

ISSN:0749-503X    

DOI:10.1002/yea.320101010

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractDNA was isolated from cells ofSaccharomyces cerevisiaeincubated under conditions that enriched for DNA replication intermediates. A novel form of the 2 μm plasmid was detected, in which two monomeric or dimeric circles were joined by a linear double‐stranded segment of variable length. We suggest that this molecule is a consequence of site‐specific recombination within a dimeric DNA molecule during DNA replication. The existence of this molecule provides supporting physical evidence for a variant of the model of 2 μ plasmid amplification first proposed by Futcher (

ISSN:0749-503X    

DOI:10.1002/yea.320101011

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe describe a rapid, sensitive and semi‐quantitative plate assay for monitoring pheromone activity in the fission yeastSchizosaccharomyces pombe. It is based on the observation that meiosis requires stimulation by pheromone and exploits diploid strains that will only sporulate after addition of exogenous pheromone. The tester strains are heterozygous for mating type, are non‐switching, and are mutated in one of the early subfunctions (eithermat1‐Mcormat1‐Pc), so that meiosis is only induced after exposure to exogenous pheromone (M‐factor or P‐factor, respectively). Pheromone activity is assessed as an iodine‐positive halo of sporulation surrounding the pheromone source, and the width of the halo is related to the amount of pheromone being produced. The assay is sufficiently sensitive to monitor the low amount of M‐factor produced by anM mam1strain, and its sensitivity towards P‐factor is greatly increased by using a hyper‐sensitive tester strain lacking the Sxa2 protease that is believed to degrade this pheromone. We also demonstrate that the production of P‐factor is very much stimulated by exposure

ISSN:0749-503X    

DOI:10.1002/yea.320101012

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY