摘要:

AbstractUsing the anticoagulant, hirudin, from the leechHirudo medicinalisas a secreted reporter protein, the influence of physiological parameters on activity and regulation of the yeast (Saccharomyces cerevisiae) metallothionein (CUP1) promoter was studied. Induction ofCUP1‐directed hirudin expression from 2μ‐based vectors was possible at any time point during diauxic batch growth, even in cells approaching stationary phase. The highest titers of hirudin were obtained when theCUP1promoter was activated immediately following inoculation of the cultures. If such a pseudo‐constitutive fermentation strategy was adopted, the promoter was superior to an optimized variant (GAPFL) of the strong, constitutive GAPDH promoter. This superiority was primarily due to the relative independence ofCUP1promoter activity of the physiological status of host cells: whilst the maximal strength of theCUP1and GAPFL promoters was comparable,CUP1‐directed hirudin expression was high in all phases of diauxic batch growth, whereas hirudin production from the GAPFL promoter declined in post‐diauxic cultures. High activity of theCUP1promoter was observed on both a fermentable (glucose) and a non‐fermentable (ethanol) carbon source. Hirudin expression could be adjusted to different levels by varying the amount of inducer (cupric sulphate) added to cultures. The copper concentrations required for maximal promoter induction had no negative effects on host growth and interfered with neither hirudin secretion nor with the biological activity of the peptide. Overexpression of the transcriptional activator, ACE1, resulted in increased levels of hirudin mRNA. Hirudin titers increased in parallel to mRNA concentrations in cultures grown in the presence of low concentrations of copper. In contrast, at high copper doses, elevated levels of the ACE1 protein resulted in inferior hirudin production. Cells overexpressingACE1while harbouring aCUP1‐drived hirudin expression cassette showed slow growth and poor plasmid maintenance. It was tested whether this might be the result of a block in the secretory pathway; however, measurements of intracellular hirudin did not support this hypothesis. The data rather indicated that hirudin production was limited by a metabolic constraint downstream of transcription but upstream of the sec

ISSN:0749-503X    

DOI:10.1002/yea.320100302

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe location and sequence of thePDA1gene, encoding the E1α subunit of the pyruvate dehydrogenase (PDH) complex fromSaccharomyces cerevisiae, were determined. ThePDA1gene was located on a 6·2 kb fragment of chromosome V, approximately 18 kb centromere distal toRAD3. Consistent with this, thePDA1gene was genetically mapped at 4 cM fromRAD3. A part of the 6·2 kb fragment of chromosome V was sequenced. The nucleotide sequence contained thePDA1open reading frame and the entire putative promoter. Computer analysis revealed a putative GCN4 binding motif in thePDA1promoter. The presence of transcriptional elements was experimentally determined by deletion analysis. To this end, ExoIII deletions were constructed in the 5′ to 3′ direction of thePDA1promoter and effects on transcription were determined by Northern analysis. Transcription was unaffected upon deletion to position − 190 relative to the ATG start codon. Deletions from position − 148 and beyond, however, reduced promoter activity at least 40‐fold. Apparently the 42 bp between nucleotides − 190 and − 148 contain an element essential for transcription. Inactivation of thePDA1promoter could not be attributed to deletions of a recognizable TATA element or any known yeast regulatory motifs. The possible role of the CCCTT sequence present in the 42 bp region and also in the promoters of the other genes encoding subunits of the PDH comp

ISSN:0749-503X    

DOI:10.1002/yea.320100303

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractA 3·6 kb DNA fragment fromSaccharomyces douglasii, containing theARG4gene, has been cloned, sequenced and compared to the corresponding region fromSaccharomyces cerevisiae. The organization of this region is identical in both yeasts. It contains besides theARG4gene, another complete open reading frame (ORF) (YSD83) and a third incomplete one (DED81). TheARG4and theYSD83coding regions differ from theirS. cerevisiaehomologs by 8.1% and 12·5%, respectively, of base substitutions. The encoded proteins have evolved differently: amino acid replacements are significantly less frequent in Arg4 (2·8%) than in Ysc83 (12·4%) and most of the changes in Arg4 are conservative, which is not the case for Ysc83. The non‐coding regions are less conserved, with small AT‐rich insertions/deletions and 20% base substitutions. However, the level of divergence is smaller in the aligned sequences of these regions than in silent sites of the ORFs, probably revealing a higher degree of constraints. The Gcn4 binding site and the region where meiotic double‐strand breaks occur, are fully conserved. The data confirm that these two yeasts are evolutionarily closely related and that comparisons of their sequences might reveal conserved protein and DNA domains not expected to be found in sequence comparisons between more diverged

ISSN:0749-503X    

DOI:10.1002/yea.320100304

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have developed a procedure to purify nucleosomal assembly‐competent histones as a mixture of H2A, H2B, H3 and H4 from isolated nuclei of the yeastSaccharomyces cerevisiaewith a purity of 70–80%. The mixture contained each of the histone subunits approximately at the equi‐molar ratio. Plasmid pBR322 DNA was assembled into nucleosomes with the purified yeast histones in the presence of nucleoplasmin from unfertilized eggs of the frogXenopus laevis. The efficiency of assembly of yeast histones was comparable to that of core histones purified from HeLa cells. The length of DNA fragment wrapping around a core histone particle and the molar ratio of histone components in an assembled nucleosome particle were estimated to be 150 ± 10 bp long and H2A:H2B:H3:H4 = 1·0:0·9:0·9:1·0, re

ISSN:0749-503X    

DOI:10.1002/yea.320100305

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractCystathionine β‐synthase (β‐CTSase), which catalyses cystathionine synthesis from serine and homocysteine, was purified to homogeneity fromSaccharomyces cerevisiae. The molecular mass of the enzyme was estimated to be 235 kDa by gel filtration and 55 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, indicating that it is a homotetramer. The N‐terminal amino acid sequence of the enzyme perfectly coincided with that deduced from the nucleotide sequence ofCYS4, except for the absence of initiation methionine. The purified β‐CTSase catalysed cysteine synthesis from serine (orO‐acetylserine) and H2S. From this finding, we discuss the multifunctional nature and evolutionary divergence of S‐meta

ISSN:0749-503X    

DOI:10.1002/yea.320100306

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have identified three yeast genes,KES1,HES1andOSH1, whose products show homology to the human oxysterol binding protein (OSBP). Mutations in these genes resulted in pleiotropic sterol‐related phenotypes. These include tryptophan‐transport defects and nystatin resistance, shown by double and triple mutants. In addition, mutant combinations showed small but apparently cumulative reductions in membrane ergosterol levels. The three yeast genes are also functionally related as overexpression ofHES1orKES1alleviated the tryptophan‐transport defect inkes1Δorosh1Δmutants, respectively. Our study implicates this new yeast gene family in ergosterol synthesis and provides comparative evidence of a role for human OSBP in cholesterol sy

ISSN:0749-503X    

DOI:10.1002/yea.320100307

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have used four glycoproteins as markers to study how disulfide bond formation and protein folding effect the intracellular transport of proteins in yeast. Under normal conditions, the vacuolar enzyme carboxypeptidase Y (CPY) and the secretory stress‐protein hsp150 acquired disulfide bonds in the endoplasmic reticulum (ER). Treatment of living cells with the reducing agent dithiothreitol (DTT) prevented disulfide formation of newly synthesized CPY and hsp150, resulting in retention of the proteins in the ER. When DTT was removed, the sulfhydryls were reoxidized, and the transport of the proteins to their correct destinations was resumed. Even mature CPY, located in the vacuole, could be reduced with DTT, and reoxidized after removal of the drug. DTT treatment blocked intracellular transport of hsp150 only when present during the synthesis and translocation of the protein. Reduction of folded hsp150, accumulated in the ER due to asecblock prior to DTT treatment, did not inhibit its secretion. The Kar2p/BiP protein, a component of the ER lumen, was found to be associated with fully translocated reduced hsp150, but not with native hsp150, suggesting that Kar2p/BiP may be involved in the putative retention mechanism. The cysteine‐free pro‐α‐factor, and invertase which was shown to have free sulfhydryls, were secreted and modified similarly in the presence and absence of DTT, showing that the secretory pathway of yeast functioned under reducing co

ISSN:0749-503X    

DOI:10.1002/yea.320100308

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have clonedNES24using a temperature‐sensitivenes24‐1mutant as a host and sequenced a 3162 bpXhoI‐EcoRI DNA fragment containing theNES24gene. Computer analysis revealed that this segment contains a 1806 bp open reading frame which is needed for complementation of thenes24‐1mutation. We foundSUP8in the region upstream of theNES24gene, placing theNES24gene on chromosome XIII. A protein homology search indicated thatNES24encodes a new protein. The disruption of theNES24gene resulted in temperature‐sensitive growth. The sequence has been deposited in DDBJ/EmBL/GenBank data bases under Accession Numb

ISSN:0749-503X    

DOI:10.1002/yea.320100309

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractATP‐binding cassette (ABC) transporters share significant sequence identity within their ATP‐binding domains. Degenerate oligonucleotides based on highly conserved portions of the ATP‐binding domain genes were used to clone portions of two members of the ABC gene superfamily fromSaccharomyces cerevisiaeDNA. These genes were designatedMDL1andMDL2(for multidrug resistance‐like). EachMDLgene is predicted to encode a single set of transmembrane domains and a single ATP‐binding domain, thus theMDLgene products are ‘half‐molecule’ ABC proteins. The two genes were mapped to precise regions on chromosomes XII and XVI and show a considerable similarity to the mammalian P‐glycoprotein/multidrug resistance (MDR) and peptide transporter (TAP) genes. Preliminary analysis of null mutants constructed by gene replacement has indicated that theMDLgenes are not essential for viability of yeast. The sequences have been deposited in the GenBank data library under Accession Numbers L16958 (Locus YSCBCSA) and L1695

ISSN:0749-503X    

DOI:10.1002/yea.320100310

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractDuring the sequencing of the geneGSP2fromSaccharomyces cerevisiae, we have encountered an adjacent open reading frame having strong homology to the 3‐phosphoserine aminotransferase (E.C.2.6.1.52) from other organisms. In this report, we present the sequence for this yeastSERC, and evidence that its deletion from the yeast genome leads to serine dependency. The sequence has been deposited in the GenBank data library under Accession Number L2091

ISSN:0749-503X    

DOI:10.1002/yea.320100311

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY