摘要:

AbstractThebglAgene which encodes a β‐glucosidase fromBacillus polymyxa, has been expressed inSaccharomyces cerevisiaeunder control of the yeastCYC‐GALpromoter. Strains have been constructed which carry the gene in different locations: in a multicopy plasmid, a single integration at theURA3locus, or multiple integrations at theRDN1locus. Integrative transformation atRDN1yielded genetically stable clones with a high level of β‐glucosidase activity. Coordinated overexpression of the GAL4 inducer protein further increased the level of enzyme activity, although eventually caused the lysis of the cultures. Diploid, triploid and tetraploid strains derived from the transformants with multiple integrations were constructed and expression of β‐glucosidase activity in different conditions of growth was assayed. While per‐cell activity increased with ploidy, specific activity was about the same in strains of equivalent genotype regardless of ploidy. Genetically stable and regulated expression inSaccharomycesof β‐glucosidase activity is interesting for the development of strains able to ferment β‐glycosidic sugars (i.e. cellobiose and lactose). From another point of view, thebglAproduct proved to be a convenient reporter enzyme to monitor heterologo

ISSN:0749-503X    

DOI:10.1002/yea.320110502

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractGrowth efficiency and regulation of key enzyme activities were studied in carbon‐ and energy‐limited chemostat cultures ofSaccharomyces cerevisiaegrown on mixtures of glucose and ethanol at a fixed dilution rate. Biomass yields on substrate carbon and oxygen could be adequately described as the net result of growth on the single substrates. Activities of isocitrate lyase and malate synthase were not detected in cell‐free extracts of glucose‐limited cultures. However, both enzymes were present when the ethanol fraction in the reservoir medium exceeded the theoretical minimum above which the glyoxylate cycle is required for anabolic reactions. Fructose‐1,6‐bisphosphatase activity was only detectable at high ethanol fractions in the feed, when activity of this enzyme was required for synthesis of hexose phosphates. Phospho‐enol‐pyruvate‐carboxykinase activity was not detectable in extracts from glucose‐grown cultures and increased with the ethanol fraction in the feed. It is concluded that, during carbon‐limited growth ofS. cerevisiaeon mixtures of glucose and ethanol, biosynthetic intermediates with three or more carbon atoms are preferentially synthesized from glucose. Synthesis of the key enzymes of gluconeogenesis and the glyoxylate cycle is adapted to the cells′ requirement for these intermediates. The gluconeogenic enzymes and their physiological antagonists (pyruvate kinase, pyruvate carboxylase and phosphofructokinase) were expressed simultaneously at high ethanol fractions in the feed. If futile cycling is prevented under these conditions, this is not primarily achieved by tight cont

ISSN:0749-503X    

DOI:10.1002/yea.320110503

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe have isolated a genomic DNA fragment that complements the yeast temperature‐sensitivecytmutation, causing respiratory deficiency and accumulation of porphyrins (Sugimuraet al., 1966). Partial DNA sequencing of the complementing region and search for similarity in the DNA and protein databases revealed that (1) the gene had been previously isolated by complementation of the mutationts2326(Langgutet al., 1986; accession number X04694), and (2) it encodes a protein with 18–23% identity to uroporphyrinogen III synthases from different sources. This enzyme catalyses the fourth step in the heme biosynthetic pathway and we named its geneHEM4. Ahem4Δ disruption mutation was constructed which had phenotypes identical to thecytmutation. Biochemical analysis confirmed the absence of uroporphyrinogen III synthase activity in bothhem4Δ andcytmutant st

ISSN:0749-503X    

DOI:10.1002/yea.320110504

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractTheURA5gene ofYarrowia lipolyticaencoding the orotate phosphoribosyl transferase (OPRTase, EC2.4.2.10) was isolated by target integration in a mutant strain originally namedura2.21. The nucleotide sequence of the gene predicts a protein with high similarities with the OPRTases fromSaccharomyces cerevisiae, Podospora anserinaandEscherichia coliand to a lesser extent with that ofDictyostelium discoideum. The transcription start point has been mapped by primer extension analysis and indicates the existence of a long leader sequence in the corresponding mRNA. Northern‐blot hybridization revealed theURA5transcript to be approximately 0·94 kb. Deletion of theURA5gene inY. lipolyticaproduced a leaky phenotype similar to the one described for theura5mutation inS. cerevisiae. TheURA5gene ofY. lipolyticawas able to complement functionally theura5mutation ofS. cerevisiae. The sequence presented here has been submitted to the EMBL data library under Accession Number Z225

ISSN:0749-503X    

DOI:10.1002/yea.320110505

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractNorthern analysis showed that DNA from the flocculation geneFLO1hybridized to mRNA molecules of 4·8 kb. This transcript was specific for theFLO1gene at the right end of chromosome I since disruption of this gene resulted in the disappearance of the transcript. We further found an absolute correlation between flocculation and the presence of transcripts hybridizing toFLO1DNA, both in various flocculent and non‐flocculent strains and in cells from the non‐flocculating and flocculating stages of growth. In all cases transcripts were present in flocculating and absent from non‐flocculating cultures. From these results we conclude that theFLO1gene is transcriptionally regulated.Mutations inTUP1orSSN6cause flocculation. Several transcripts hybridizing toFLO1DNA were present in the mutants but not in the corresponding wild‐type strains. Disruption of theFLO1gene in thetup1andssn6strains showed that one of the transcripts corresponded to theFLO1gene. Disruption ofFLO1did not abolish flocculation completely but only reduced it, indicating that at least two flocculation genes, includingFLO1, are activated or derepressed by mutations in theTUP1/SSN6regulatory

ISSN:0749-503X    

DOI:10.1002/yea.320110506

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractPreviously, a 38‐base‐pair (bp) region in the 3′ untranslated portion of theSaccharomyces cerevisiaeiso‐1‐cytochromecgene, was shown to be required for both normalCYC1mRNA 3′ end formation (Zaret and Sherman, 1982), and efficient transcription termination (Russo and Sherman, 1989). In another study, specific sequences such as TATATA, TACATA, and TAGTAGTA were shown to be involved in mRNA 3′ end formation inS. cerevisiae(Russoet al., 1991). In this report, anin vivoplasmid stability assay has been utilized to show that these and related sequences are also involved in transcription termination, at varying efficiencies, and in an orientation‐dependent manner. For example: the sequence TATATA appeared to terminate transcription almost as efficiently as the original wild type 38‐bp region; whereas, the sequences TAGATATATGTAA and TACATA were less efficient, and TTTTTTTATA had little, if any, transcription termination function. In contrast, none of these sequences appeared to terminate transcription in the reverse orientation. Therefore, it appears that certain sequence signals capable of promoting mRNA 3′ end formation in yeast, are also directly involved in transc

ISSN:0749-503X    

DOI:10.1002/yea.320110507

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe sequence of a 5653 bp DNA fragment of the right arm of chromosome II ofSaccharomyces cerevisiaecontains two unknown open reading frames (YBR1212 and YBR1213) next to geneCDC28. Gene disruption reveals both putative genes as non‐essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Sur1 protein ofS. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with aLactococcus lactis‐secreted 45 kDa protein and 24% identity with theSaccharomyces cerevisiae AGA1gene product. The total sequence of the fragment has been submitted to the EMBL databank (accession number X802

ISSN:0749-503X    

DOI:10.1002/yea.320110508

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractA 6·6 kb genomic DNA fragment from the yeastKluyveromyces lactiswas isolated. Sequence analysis of this fragment revealed the presence of two incomplete open reading frames (ORFs) in one strand, one coding for the carboxyl terminus of the plasma membrane H+‐ATPase and the other for the amino terminus of an unidentified product. In the complementary strand, a full‐length ORF which encodes for a protein homologous to the yeast NADPH‐dependent Old Yellow Enzyme was found. The deduced amino acid sequence of this ORF predicts a protein of 398 residues with 84% similarity in its full length to OYE1 fromSaccharomyces carlsbergensisand OYE2 fromSaccharomyces cerevisiae. In addition, an internal region showed considerable similarity to the bile acid‐inducible polypeptide fromEubacterium sp., to the NADH oxidase fromThermoanaerobium brockii, to the trimethylamino dehydrogenase from bacterium W3A1and to the estrogen‐binding protein fromCandida albicans, suggesting a functional or structural relationship between them. Inactivation of theKYE1(KluyveromycesYellowEnzyme) gene by deletion of 0·6 kb fragment between positions +358 and +936 produced viable cells with a slight increase in their generation time. Haploid cells carrying the disrupted allele showed one‐third of the NADPH oxidase activity, compared to wild‐type cells. Southern blotting analysis of digested DNA and chromosomes separated by contour‐clamped homogeneous electric field electrophoresis fromK. lactisindicated that this is a single‐copy gene and it is localized on chromosome II, whose molecular size has been estimated to be approximately 1·3 Mb. The sequence reported in this paper has been deposited in the GenBank data base (A

ISSN:0749-503X    

DOI:10.1002/yea.320110509

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractA gene that complements theleu2mutation ofSaccharomyces cerevisiaehas been cloned fromSchwanniomyces occidentalis. The gene codes for a protein of 379 amino acids. As expected for aSchwanniomycesgene, it has a high AT content, which is also reflected in the codon usage. The sequence homology with other knownleu2complementing genes is low. The nucleotide sequence of theSchw. occidentalis LEU2gene has been assigned the Accession Number X79823 SOLEU2 by EMBL.

ISSN:0749-503X    

DOI:10.1002/yea.320110510

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractIn the course of the European yeast genome sequencing project, we determined 23,920 bp of a continuous chromosome II right arm sequence. Analysis of data revealed 13 open reading frames (ORFs), three of which corresponded to previously identified genes; two tRNA genes and one repetitive element. One ORF showed considerable homology (46%) to a hypothetical chromosome III gene; another, putatively very hydrophobic gene product, was 30% identical to the heat‐shock protein HSP30. Two ORFs were homologous to human genes. The complete sequence was submitted to the EMBL data bank under the Accession Number Z46260 Authorin submission

ISSN:0749-503X    

DOI:10.1002/yea.320110511

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY