Biomedical subjects

I S Pretorius

Publications and source records attributed to I S Pretorius.

At least 37 records · Page 2Linked to original sources

PubMed1999

Msn1p/Mss10p, Mss11p and Muc1p/Flo11p are part of a signal transduction pathway downstream of Mep2p regulating invasive growth and pseudohyphal differentiation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a network of signal transduction pathways governs the switch from yeast-type growth to pseudohyphal and invasive growth that occurs in response to nutrient limitation. Important elements of this network have been identified, including nutrient signal receptors, GTP-binding proteins, components of the pheromone-dependent MAP kinase cascade and several transcription factors. However, the structural and functional mapping of these pathways is far from complete. Here, we present data regarding three genes, MSN1/MSS10, MSS11 and MUC1/FLO11, which form an essential part of the signal transduction network establishing invasive growth. Both MSN1 and MSS11 are involved in the co-regulation of starch degradation and invasive growth. Msn1p and Mss11p act downstream of Mep2p and Ras2p and regulate the transcription of both STA2 and MUC1. We show that MUC1 mediates the effect of Msn1p and Mss11p on invasive growth. In addition, our results suggest that the activity of Msn1p is independent of the invasive growth MAP kinase cascade, but the Mss11p is required for the activation of pseudohyphal and invasive growth by Ste12p. We also show that starch metabolism in S. cerevisiae is subject to regulation by components of the MAP kinase cascade.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

Divergent regulation of the evolutionarily closely related promoters of the Saccharomyces cerevisiae STA2 and MUC1 genes.

The 5' upstream regions of the Saccharomyces cerevisiae glucoamylase-encoding genes STA1 to -3 and of the MUC1 (or FLO11) gene, which is critical for pseudohyphal development, invasive growth, and flocculation, are almost identical, and the genes are coregulated to a large extent. Besides representing the largest yeast promoters identified to date, these regions are of particular interest from both a functional and an evolutionary point of view. Transcription of the genes indeed seems to be dependent on numerous transcription factors which integrate the information of a complex network of signaling pathways, while the very limited sequence differences between them should allow the study of promoter evolution on a molecular level. To investigate the transcriptional regulation, we compared the transcription levels conferred by the STA2 and MUC1 promoters under various growth conditions. Our data show that transcription of both genes responded similarly to most environmental signals but also indicated significant divergence in some aspects. We identified distinct areas within the promoters that show specific responses to the activating effect of Flo8p, Msn1p (or Mss10p, Fup1p, or Phd2p), and Mss11p as well as to carbon catabolite repression. We also identified the STA10 repressive effect as the absence of Flo8p, a transcriptional activator of flocculation genes in S. cerevisiae.

DNA-Binding Proteins↗

PubMed1998

Engineering yeast for efficient cellulose degradation.

Saccharomyces cerevisiae produces several beta-1,3-glucanases, but lacks the multicomponent cellulase complexes that hydrolyse the beta-1,4-linked glucose polymers present in cellulose-rich biomass as well as in haze-forming glucans in certain wines and beers. We have introduced into S. cerevisiae a functional cellulase complex for efficient cellulose degradation by cloning the Endomyces fibuliger cellobiase (BGL1) gene and co-expressing it with the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase (END1), the Phanerochaete chrysosporium cellobiohydrolase (CBH1) and the Ruminococcus flavefacies cellodextrinase (CEL1) gene constructs in this yeast. The END1, CBH1 and CEL1 genes were inserted into yeast expression/secretion cassettes. Expression of END1, CBH1 and CEL1 was directed by the promoter sequences derived from the alcohol dehydrogenase II (ADH2), the phosphoglycerate kinase I (PKG1) and the alcohol dehydrogenase I (ADH1) genes, respectively. In contrast, BGL1 was expressed under the control of its native promoter. Secretion of End1p and Cel1p was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1), whereas Cbh1p and Bgl1p were secreted using their authentic leader peptides. The construction of a fur1 ura3 S. cerevisiae strain allowed for the autoselection of this multicopy URA3-based plasmid in rich medium. S. cerevisiae transformants secreting biologically active endo-beta-1,4-glucanase, cellobiohydrolase, cellodextrinase and cellobiase were able to degrade various substrates including carboxymethylcellulose, hydroxyethylcellulose, laminarin, barley glucan, cellobiose, polypectate, birchwood xylan and methyl-beta-D-glucopyranoside. This study could lead to the development of industrial strains of S. cerevisiae capable of converting cellulose in a one-step process into commercially important commodities.

Bacterial Proteins↗

PubMed1998

Identification of a functional TATA element in the STA2 glucoamylase gene promoter from Saccharomyces cerevisiae.

Accurate transcription by RNA polymerase II is usually dependent on the presence of a TATA element, and/or an initiator element, in the promoters of protein-encoding genes. The STA1-3 genes, encoding three glucoamylase isozymes (Sta1p, Sta2p and Sta3p, respectively) responsible for starch hydrolysis in the yeast Saccharomyces cerevisiae, have been shown to contain long and complex promoters with several regulatory regions. These promoters are also virtually identical to the yeast MUC1 gene promoter; this gene encodes a mucin-like protein and is evolutionary linked to, and transcriptionally co-regulated with, STA1-3. The STA1-3 genes contain two putative TATA sequences; one conforming to the typical TATA box sequence, TATAAA, and another with the sequence of TATAAT. Here we present a study into the functional relevance of these putative TATA sequences and their effects on the transcription of the STA2 gene (as a representative model of the STA1-3 multigene family) and, by analogy, the MUC1 gene. We show that the TATAAA motif is the functional TATA box for STA2 and influences transcript levels, transcript initiation sites, and glucoamylase activities.

Enzyme Activation↗

PubMed1998

Genetic and fermentation properties of the K2 killer yeast, Saccharomyces cerevisiae T206.

Saccharomyces cerevisiae T206 K+R+, a K2 killer yeast, was differentiated from other NCYC killer strains of S. cerevisiae on the basis of CHEF-karyotyping and mycoviral RNA separations. Genomic DNA of strain T206 was resolved into 13 chromosome bands, ranging from approximately 0.2 to 2.2 Mb. The resident virus in strain T206 yielded L and M RNA species of approximately 5.1 kb and 2.0 kb, respectively. In micro-scale vinifications, strain T206 showed a lethal effect on a K-R- mesophilic wine yeast. Metabolite accumulation and toxin activity were measured over a narrow pH range of 3.2 to 3.5. Contrary to known fermentation trends, the challenged fermentations were neither stuck nor protracted although over 70% of the cell population was killed. Toxin-sensitive cells showed cytosolic efflux.

Acetic Acid↗

PubMed1997

Over-expression of the Saccharomyces cerevisiae exo-beta-1,3-glucanase gene together with the Bacillus subtilis endo-beta-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene in yeast.

The EXG1 gene encoding the main Saccharomyces cerevisiae exo-beta-1,3-glucanase was cloned and over-expressed in yeast. The Bacillus subtilis endo-1,3-1,4-beta-glucanase gene (beg1) and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene (end1) were fused to the secretion signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S) and inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T). Constructs ADH2P-MF alpha 1S-beg1-ADH2T and ADH2P-MF alpha 1S-end 1-ADH2T designated BEG1 and END1, respectively, were expressed separately and jointly with EXG1 in S. cerevisiae. The construction of fur 1 ura3 S. cerevisiae strains allowed for the autoselection of these multicopy URA3-based plasmids in rich medium. Enzyme assays confirmed that co-expression of EXG1, BEG1 and END1 enhanced glucan degradation by S. cerevisiae.

Amino Acid Sequence↗

PubMed1997

Cloning of the Bacillus pumilus beta-xylosidase gene (xynB) and its expression in Saccharomyces cerevisiae.

A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the beta-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique. The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T) sequences on a multicopy episomal plasmid (pDLG11). The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S) before insertion between the ADH2P and ADH2T sequences on a similar multicopy episomal plasmid (pDLG12). The resulting construct ADH2P-MF alpha 1S-xynB-ADH2T was designated XLO1. Both plasmids pDLG11 and PDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional beta-xylosidase. The total beta-xylosidase activity remained cell-associated with a maximum activity of 0.09 nkat/ml obtained when the recombinant S. cerevisiae strain was grown for 143 h in synthetic medium. The temperature and pH optima of the recombinant Xlo1 enzyme were 45-50 degrees C and pH 6.6 respectively. The enzyme was thermostable at 45 degrees C; however, at 60 degrees C most of the Xlo1 was inactive after 5 min.

Bacillus↗

PubMed1997

MSS11, a novel yeast gene involved in the regulation of starch metabolism.

Expression of the STA1-3 glucoamylase genes, responsible for starch degradation in Saccharomyces cerevisiae, is down regulated by the presence of STA10. In order to elucidate the role of STA10 in the regulation of the glucoamylase system, a multicopy genomic library was constructed and screened for genes that enhanced growth of a STA2-STA10 S. cerevisiae strain on starch media. This screen allowed us to clone and characterize a novel activator gene of STA2 (and by extrapolation, STA1 and STA3), designated MSS11. A strain transformed with multiple copies of MSS11 exhibits increased levels of STA2 mRNA and, consequently, increased glucoamylase activity. Deletion of MSS11, located on chromosome XIII, results in media-dependent absence of glucoamylase synthesis. MSS11 has not been cloned previously and the encoded protein, Mss11p, is not homologous to any other known protein. An outstanding feature of Mss11p is that the protein contains regions of 33 asparagine residues interrupted by only three serine residues, and 35 glutamine residues interrupted by a single histidine residue. Epistasis studies showed that deletion of MSS11 abolishes the activation of STA2 caused by the over-expression of MSS10, a previously identified gene. In turn, it was found that deletion of MSS10 still allows activation of STA2 by over-expression of MSS11. Mss11p therefore appears to be positioned below Mss10p in a signal transduction pathway.

Amino Acid Sequence↗

PubMed1997

Coregulation of starch degradation and dimorphism in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae, the exemplar unicellular eukaryote, can only survive and proliferate in its natural habitats through constant adaptation within the constraints of a dynamic ecosystem. In every cell cycle of S. cerevisiae, there is a short period in the G1 phase of the cell cycle where "sensing" transpires; if a sufficient amount of fermentable sugars is available, the cells will initiate another round of vegetative cell division. When fermentable sugars become limiting, the yeast can execute the diauxic shift, where it reprograms its metabolism to utilize nonfermentable carbon sources. S. cerevisiae can also initiate the developmental program of pseudohyphal formation and invasive growth response, when essential nutrients become limiting. S. cerevisiae shares this growth form-switching ability with important pathogens such as the human pathogen, Candida albicans, and the corn smut pathogen Ustilago maydis. The pseudohyphal growth response of S. cerevisiae has mainly been implicated as a means for the yeast to search for nutrients. An important observation made was that starch-degrading S. cerevisiae strains have the added ability to form pseudohyphae and grow invasively into a starch-containing medium. More significantly, it was also shown that the STA1-3 genes encoding three glucoamylase isozymes responsible for starch hydrolysis in S. cerevisiae are coregulated with a gene, MUC1, essential for pseudohyphal and invasive growth. At least two putative transcriptional activators, Mss10p and Mss11p, are involved in this regulation. The Muc1p is a putative integral membrane-bound protein similar to mammalian mucin-like proteins that have been implicated in the ability of cancer cells to invade other tissues. This provided us with an excellent example of integrative control between nutrient sensing, signaling, and differential development.

Adaptation, Physiological↗

PubMed1996

Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast.

Pseudohyphal differentiation in Saccharomyces cerevisiae was first described as a response of diploid cells to nitrogen limitation. Here we report that haploid and diploid starch-degrading S. cerevisiae strains were able to switch from a yeast form to a filamentous pseudohyphal form in response to carbon limitation in the presence of an ample supply of nitrogen. Two genes, MSS10 and MUC1, were cloned and shown to be involved in pseudohyphal differentiation and invasive growth. The deletion of MSS10 resulted in extremely reduced amounts of pseudohyphal differentiation and invasive growth, whereas the deletion of MUC1 abolished pseudohyphal differentiation and invasive growth completely. Mss10 appears to be a transcriptional activator that responds to nutrient limitation and coregulates the expression of MUC1 and the STA1-3 glucoamylase genes, which are involved in starch degradation. MUC1 encodes a 1367-amino acid protein, containing several serine/threonine-rich repeats. Muc1 is a putative integral membrane-bound protein, similar to mammalian mucin-like membrane proteins that have been implicated to play a role in the ability of cancer cells to invade other tissues.

Amino Acid Sequence↗

PubMed1996

Cloning, mapping and characterization of a genomic copy of the Lipomyces kononenkoae alpha-amylase-encoding gene (LKA1).

The expression in Saccharomyces cerevisiae and Schizosaccharomyces pombe of a cDNA copy of the Lipomyces kononenkoae IGC4052B alpha-amylase gene (LKA1), linked to the phosphoglycerate kinase gene (PGK1) promoter, resulted in the extracellular production of biologically active alpha-amylase (LKA1). However, transformation of S. cerevisiae and Schiz. pombe with a cosmid clone containing the complete genomic copy of LKA1, expressed from its native promoter, did not result in secretion of active alpha-amylase by any of the transformants. When the cDNA copy of LKA1 was expressed in S. cerevisiae under control of the wild-type L, kononenkoae promoter, biologically active alpha-amylase was secreted into the culture medium, indicating the recognition of the LKA1 promoter in S. cerevisiae. Sequence analysis of the GC-rich LKA1 promoter revealed canonical sequences that are homologous to the TATAAA, CAAT and CCAAT boxes and GCN4-binding sites that are present in several promoter sequences of S. cerevisiae. Primer extension analysis of LKA1 transcripts in L. kononenkoae indicated major initiation sites at nucleotides -64 and -65. S. cerevisiae and Schiz. pombe cells transformed with a plasmid containing the open reading frame of the genomic copy of LKA1, linked to the PGK1 promoter, did not produce alpha-amylase. Polymerase chain reaction mapping and sequence analysis revealed the presence of a 61-bp intron in the genomic copy of LKA1 that impaired synthesis of biologically active alpha-amylase in S. cerevisiae and Schiz. pombe. This intron contains donor, acceptor and branch sequences that correlate with the consensus sequences identified in the introns of split genes from Schiz. pombe and mammals. Pulsed-field gradient gel electrophoresis resolved at least eight chromosomal DNAs for L. kononenkoae IGC4052B and chromoblot analysis indicated that LKA1 is located on the second smallest chromosome, designated chromosome II.

Ascomycota↗

PubMed1996

Cloning and expression of the alpha-L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae.

First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the alpha-L-arabinofuranosidase gene (abfB) was amplified with the polymerase chain reaction technique. The abfB DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1P) and terminator (PGK1T) sequences on a multicopy episomal plasmid. The resulting construct PGK1P-abfB-PGK1T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional alpha-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf2) that is 94% identical to the alpha-L-arabinofuranosidase B of A. niger N400. Maximum alpha-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively.

Amino Acid Sequence↗

PubMed1996

Co-expression of a Phanerochaete chrysosporium cellobiohydrolase gene and a Butyrivibrio fibrisolvens endo-beta-1,4-glucanase gene in Saccharomyces cerevisiae.

A cDNA fragment encoding the Phanerochaete chrysosporium cellobiohydrolase (cbh1-4) was amplified and cloned with the aid of the polymerase chain reaction (PCR) technique. The cbh1-4 gene and the Butyrivibrio fibrisolvens endo-beta-1,4-glucanase (end1) gene were successfully expressed in Saccharomyces cerevisiae under the control of the phosphoglycerate kinase-I (PGK1) and alcohol dehydrogenase-II (ADH2) gene promoters and terminators, respectively. The native P. chrysosporium signal sequence mediated secretion of cellobiohydrolase in S. cerevisiae, whereas secretion of the endo-beta-1,4-glucanase was directed by the signal sequence of the yeast mating pheromone alpha-factor (MFalpha1S). These constructs, designated CBH1 and END1, respectively, were expressed separately and jointly in S. cerevisiae. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of these multicopy URA3-based plasmids in rich medium. Enzyme assays confirmed that co-expression of CBH1 and END1 synergistically enhanced cellulose degradation by S. cerevisiae.

Agaricales↗

PubMed1996

Expression of a Trichoderma reesei beta-xylanase gene (XYN2) in Saccharomyces cerevisiae.

The XYN2 gene encoding the main Trichoderma reesei QM 6a endo-beta-1,4-xylanase was amplified by PCR from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 699-bp open reading frame that encodes a 223-amino-acid propeptide. The XYN2 gene, located on URA3-based multicopy shuttle vectors, was successfully expressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II (ADH2) and phosphoglycerate kinase (PGK1) gene promoters and terminators, respectively. The 33-amino-acid leader peptide of the Xyn2 beta-xylanase was recognized and cleaved at the Kex2-like Lys-Arg residues, enabling the efficient secretion and glycosylation of the heterologous beta-xylanase. The molecular mass of the recombinant beta-xylanase was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 27 kDa. The construction of fur1 ura3 S. cerevisiae strains allowed for the autoselection of the URA3-based XYN2 shuttle vectors in nonselective complex medium. These autoselective S. cerevisiae strains produced 1,200 and 160 nkat of beta-xylanase activity per ml under the control of the ADH2 and PGK1 promoters in rich medium, respectively. The recombinant enzyme showed highest activity at pH 6 and 60 degrees C and retained more than 90% of its activity after 60 min at 50 degrees C.

Amino Acid Sequence↗

PubMed1996

A multicopy suppressor gene, MSS10, restores STA2 expression in Saccharomyces cerevisiae strains containing the STA10 repressor gene.

Transcription of the three unlinked, homologous STA1-3 glucoamylase-encoding genes, involved in starch degradation by Saccharomyces cerevisiae, was previously shown to be down-regulated by the presence of STA10, acting via three upstream repression sequence regions that were identified in the STA2 promoter. Here we report the cloning and characterization of a putative transcriptional activator gene, MSS10 (multicopy suppressor of STA10), which, when present in multiple copies, overcomes STA10 repression. Deletion of MSS10, located on chromosome XV, resulted in media-specific extinction of glucoamylase synthesis. The nucleotide sequence of MSS10 is identical to three other genes from S. cerevisiae identified as: FUP1, a gene that enhances iron-limited growth; PHD2, a gene identified for its ability to induce pseudohyphal growth in diploid cells grown on nitrogen-limited media; and MSN1, a gene encoding a transcriptional activator involved in invertase regulation.

Cloning, Molecular↗

PubMed1995

Cloning, sequence analysis and expression in yeasts of a cDNA containing a Lipomyces kononenkoae alpha-amylase-encoding gene.

The yeast Lipomyces kononenkoae (Lk) secretes a highly active raw starch-degrading alpha-amylase (alpha Amy) that liberates reducing groups from glucose polymers containing both alpha-1,4 and alpha-1,6 bonds. The LKA1 gene encoding this industrially important alpha Amy was cloned as a 2261-bp cDNA fragment from a glucose-derepressed mutant (IGC4052B) of Lk and characterized. The nucleotide (nt) sequence of the cDNA fragment was determined, revealing an open reading frame of 1872 bp, encoding a 596 amino-acid (aa) mature protein (LKA1) with a calculated M(r) of 65,706. The similarity between the aa sequence of LKA1 and those of other alpha Amy showed four common conserved regions characteristic of the alpha Amy protein family: (A) 264DIVVNH269, (B) 349GLRIDTVKH357, (B') 376GEVFD380 and (C) 439FLENQD444. The deduced aa sequence revealed significant homology to the aa sequences of the Aspergillus oryzae, Schwanniomyces occidentalis and Saccharomycopsis fibuligera alpha Amy, various bacterial cyclomaltodextrin glucanotransferases, a beta-amylase and the 5'-region of a glucoamylase. LKA1 was expressed in Saccharomyces cerevisiae (Sc) under the control of the phosphoglycerate kinase (PGK1) promoter and Northern blot analysis showed the presence of a single 2.3-kb transcript. The 28-aa signal peptide of the LKA1 protein efficiently directed its secretion into the medium when expressed in Sc.

Amino Acid Sequence↗

PubMed1995

The S1, S2 and SGA1 ancestral genes for the STA glucoamylase genes all map to chromosome IX in Saccharomyces cerevisiae.

The polymorphic extracellular glucoamylase-encoding genes STA1 (chr. IV), STA2 (chr. II) and STA3 (chr. XIV), from Saccharomyces cerevisiae var. diastaticus probably evolved by genomic rearrangement of DNA regions (S1, S2 and SGA1) present in S. cerevisiae, and subsequent translocation to unlinked regions of chromosomal regions. S1, encoding a homologue to the threonine/serine-rich domain of STA glucoamylases (GAI-III), mapped to the right arm of chromosome IX. S2, encoding the hydrophobic leader peptide of GAI-III), was also mapped on the right arm of chromosome IX, next to S1, close to DAL81. The SGA1 sporulation-specific, intracellular glucoamylase-encoding gene is located on the left arm of chromosome IX, 32 kb proximal of HIS5.

Chromosome Mapping↗

PubMed1995

One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing alpha-amylase, glucoamylase and pullulanase.

A recombinant strain of Saccharomyces cerevisiae was constructed that contained the genes encoding a bacterial alpha-amylase (AMY1), a yeast glucoamylase (STA2) and a bacterial pullulanase (pulA). The Bacillus amyloliquefaciens alpha-amylase and S. cerevisiae var. diastaticus glucoamylase genes were expressed in S. cerevisiae using their native promoters and the encoded enzymes secreted under direction of their native leader sequences. In contrast, the Klebsiella pneumoniae pullulanase gene was placed under the control of the yeast alcohol dehydrogenase gene promoter (ADC1P) and secreted using the yeast mating pheromone alpha-factor secretion signal (MF alpha 1S). Transcription termination of the pullulanase gene was effected by the yeast tryptophan synthase gene terminator (TRP5T), whereas termination of the glucoamylase and alpha-amylase genes was directed by their native terminators. Pullulanase (PUL1) produced by recombinant yeasts containing ADC1P MF alpha 1S pulA TRP5T (designated PUL1) was further characterized and compared to its bacterial counterpart (PulA). The different genes were introduced into S. cerevisiae in different combinations and the various amylolytic Saccharomyces transformants compared to Schwanniomyces occidentalis. Introduction of PUL1 into a S. cerevisiae strain containing both STA2 and AMY1, resulted in 99% assimilation of starch.

Bacillus↗