A third gene (Delta9-3) encoding a fatty acid Delta9-clesaturase was isolated from the oil-producing fungus Mortierella alpina. The predicted protein of 512 aa shared 53 % sequence identity with the two fatty acid Delta9-desaturases, ole1p and ole2p, already described in this organism and contained three histidine boxes, four putative transmembrane domains and a C-terminal cytochrome b(s) fusion that are typical of most fungal membrane-bound fatty acid desaturases. However, unlike the M. alpina ole1 and o1e2 genes, the Delta9-3 ORF failed to complement the Saccharomyces cerevisiae ole1 mutation. GC-MS analysis of fatty-acid-supplemented ole1 yeast transformants containing the Delta9-3 gene indicated that this enzyme had negligible activity with endogenous palmitic acid (16: 0) as substrate and moderate activity (30-65 % desaturation) with endogenous stearic acid (18:0). Yeast transformants overexpressing any one of the three M. alpina fatty acid Delta9-desaturase genes or the S. cerevisiae OLE1 gene produced low amounts of hexacosenoic acid [26:1 (n-9)], a fatty acid that is not normally present in yeast cells. it follows that these Delta9-desaturases may also display low n-9 desaturation activity with very long-chain saturated fatty acid substrates. Conversely, high levels of desaturase in the endoplasmic reticulum membrane of these yeast transformants may increase the availability of suitable monounsaturated substrates for fatty acid elongation.

Phosphoribosyl-pyrophosphate synthetase (Prs) catalyses the synthesis of phosphoribosyl pyrophosphate (PRPP). an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1-PRS5, Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures, This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p, Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway, Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type, The fact that Delta prs1 and Delta prs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.

The PRS gene family in Saccharomyces cerevisiae consists of five genes each capable of encoding a 5-phosphoribosyl-1(alpha)-pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 or PRS3; simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3 together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions of PRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl pyrophosphate synthetase exists in vivo as multimeric complex(es).

3 Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK Genes encoding two distinct fatty acid ∆9-desaturases were isolated from strains of the oleaginous fungus Mortierella alpina. Two genomic sequences, ∆9-1 and ∆9-2, each containing a single intron, were cloned from strain CBS 528.72 while one cDNA clone, LM9, was isolated from strain CBS 210.32. The ∆9-1 gene encoded a protein of 445 aa which shared 99% identity with the LM9 gene product. These proteins also showed 40–60% identity to the ∆9desaturases (Ole1p) of other fungi and contained the three conserved histidine boxes, C-terminal cytochrome b5 fusion and transmembrane domains characteristic of endoplasmic reticulum membrane-bound ∆9-desaturases. LM9 and ∆9-1 are therefore considered to represent the same gene (ole1). The ole1 gene was transcriptionally active in all M. alpina strains tested and its function was confirmed by complementation of the Saccharomyces cerevisiae ole1 mutation. Fatty acid analysis of yeast transformants expressing the CBS 210.32 ole1 gene showed an elevated level of oleic acid (18:1) compared to palmitoleic acid (16:1), the major fatty acid component of wild-type S. cerevisiae. This indicated that the M. alpina ∆9-desaturase had a substrate preference for stearic acid (18:0) rather than palmitic acid (16:0). Genomic clone ∆9-2 (ole2) also encoded a protein of 445 aa which had 86% identity to the ∆9-1 and LM9 proteins and whose ORF also complemented the yeast ole1 mutation. The transcript from this gene could only be detected in one of the six M. alpina strains tested, suggesting that its expression may be strainspecific or induced under certain physiological conditions.

In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell.

Aggregation of Candida famata (Debaryomyces hansenii) is consistent with being a form of lectin-mediated yeast flocculation. Flocculation of C. famata is unusual in that it requires the presence of peptone, either in the growth medium or added later to harvested cells in buffer. Flocculation after peptone addition was rapid, being largely complete within 10 min. Heat-killed cells also flocculated, arguing for direct participation of peptone in the flocculation binding mechanism. Flocculent C. famata cells progressively lost the ability to flocculate when washed with EDTA. Flocculation was fully restored by peptone addition; calcium addition was without effect. C. famata cells were able to agglutinate erythrocytes in the presence or absence of peptone. Pronase E-treated yeast lost both the ability to haemagglutinate and self-flocculate. Haemagglutination was not diminished by progressive EDTA washing, suggesting that surface lectins remained present and active on the yeast cell walls. Non-flocculating C. famata cells mutually flocculated with non-flocculent Schizosaccharomyces pombe cells, shown to have surface-exposed galactose residues. Mutual flocculation was lost following treatment of C. famata with Pronase E. It was concluded that the cell wall of C. famata contains lectins enabling haemagglutination and mutual flocculation but lacks carbohydrate receptors for these lectins. This yeast self-flocculates only via bridging multi-valent carbohydrates; these being present in peptone.

Conference Article| November 01 1995 Are all four yeast PRS genes essential? ANDREW T. CARTER; ANDREW T. CARTER 1Genetics & Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK Search for other works by this author on: This Site PubMed Google Scholar FLORA BEICHE; FLORA BEICHE †Institut für Pharmakologie und Toxikologie, Universität Erlangen-Nürnberg, Universitätsstr. 22, D-91054 Erlangen, Germany Search for other works by this author on: This Site PubMed Google Scholar ARJAN NARBAD; ARJAN NARBAD 1Genetics & Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK Search for other works by this author on: This Site PubMed Google Scholar BJARNE HOVE-JENSEN; BJARNE HOVE-JENSEN ¶ Institute of Molecular Biology, Department of Biological Chemistry, 83H SØlvgade, DK-1307 Copenhagen K, Denmark Search for other works by this author on: This Site PubMed Google Scholar LILIAN M. SCHWEIZER; LILIAN M. SCHWEIZER 1Genetics & Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK Search for other works by this author on: This Site PubMed Google Scholar MICHAEL SCHWEIZER MICHAEL SCHWEIZER 1Genetics & Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (4): 621S. https://doi.org/10.1042/bst023621s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation ANDREW T. CARTER, FLORA BEICHE, ARJAN NARBAD, BJARNE HOVE-JENSEN, LILIAN M. SCHWEIZER, MICHAEL SCHWEIZER; Are all four yeast PRS genes essential?. Biochem Soc Trans 1 November 1995; 23 (4): 621S. doi: https://doi.org/10.1042/bst023621s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

Yeast flocculation involves binding of surface lectins to carbohydrate receptors on neighbouring cell walls. Brewing strains of Saccharomyces cerevisiae normally become flocculent in the stationary phase of growth. This paper presents evidence that lectins are synthesized in exponential phase, inserted into the cell wall, and activated later at the time of flocculation onset. Cycloheximide failed to prevent flocculation unless it was added in early growth; with later additions progressively larger degrees of flocculation occurred. Flocculation onset was delayed by cycloheximide but was otherwise cycloheximide insensitive. Preflocculent cells could be artificially activated to full flocculation by heat. Artificial activation of samples from growing yeast cultures confirmed the progressive synthesis of lectins throughout exponential growth. Pronase E treatment of whole cells prior to heating prevented any activation of flocculation. It was concluded that lectins were synthesized continuously from an early stage of growth and rapidly inserted into the cell wall (accessible by pronase E), where they remained inactive for up to 14 h, before being activated at flocculation onset by an as-yet unknown mechanism. It was found that lectin synthesis and activation occurred in all brewing strains tested.

A 3.5-kb DNA fragment from the Clarke and Carbon Escherichia coli genomic clone, pLC37-44, was sequenced on both strands. Part of the zwf gene, encoding glucose-6-phosphate dehydrogenase, and all of the edd and eda genes, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, respectively, of the Entner-Doudoroff pathway, were identified. These data are compared with those of Egan et al. [J. Bacteriol. 174 (1992) 4638-4646] and important differences were noted.