Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Mannan polysaccharides occur in the hemicellulose fraction of plant cell walls, either as structural polymers or as reserve carbohydrates. They are found predominantly in the seeds of leguminous plants in the form of galactomannan, and in softwoods as galactoglucomannan. Endo-I,4-I3-mannanases hydrolyze mannan polysaccharides to oligosaccharides of various lengths. These enzymes are secreted as single catalytic modules or as part of multi-modular proteins by fungi, bacteria, plants and animals. For example, the l3-mannanase of Aspergillus aculeatus, designated Aa-Man5A, is secreted as a single catalytic module, whereas that of Trichoderma reesei, designated Tr-Man5A, contains a l3-mannanase catalytic module linked to a cellulose-binding module by a Pro- Ser-Thr-rich linker. Heterologous gene expression in yeast provides the opportunity to produce individual hydrolytic enzymes in a host expression system devoid of related activities. Saccharomyces cerevisiae has a well-developed expression system and has frequently been used as a model organism for heterologous gene expression. A number of autoselection systems have been devised so that recombinant S. cerevisiae strains can be cultivated in any medium of choice without exerting selective pressure. An autoselection system based on defective chromosomal ura3 andfurl genes involved in the pyrimidine biosynthesis pathway of S. cerevisiae, and complementation of the ura3 gene with a multicopy plasmid-borne URA3 gene, were used in this study. The man1 of A. aculeatus gene encoding Aa-Man5A was cloned and expressed in autoselective S. cerevisiae under the regulation of the alcohol dehydrogenase (ADH2PT) and the phosphoglycerate kinase (PGK1PT) promoter and terminator sequences. Expression of man1 under both promoters resulted in high production levels of Aa- Man5A. The production levels were significantly higher than the levels of endo-l,4-13- mannanases produced by heterologous expression in Escherichia coli, and were comparable to the production levels of enzymes produced in Pichia pastoris, which presumably has a higher secretion capacity than S. cerevisiae. The recombinant yeast strain expressing man1 under the regulation of the PGK1p promoter displayed stunted biomass formation during the logarithmic phase, which was relieved when the native f3- mannanase secretion signal was replaced with the yeast MFuis secretion signal. The recombinant Aa-Man5A displayed biochemical properties similar to those of the native Aa-Man5A. The recombinant enzyme hydrolyzed unsubstituted mannan to predominantly mannose, mannobiose, and mannotriose. The expression of the man1 and man1 ácbd gene constructs of T reesei in S. cerevisiae fur 1::LEU2 strains under the regulation of the PGK1 PT promoter and terminator resulted in the production and secretion of Tr-Man5A and Tr-Man5A~CBD (lacking the cellulose binding module), respectively. However, the production levels of both proteins were approximately I5-fold lower than the production levels of Aa-Man5A. These levels did not improve after replacement of the native secretion signal with the MFuis secretion signal. Interestingly, reducing the cultivation temperature from 30°C to 20°C led to a five-fold increase in the secreted levels of Tr-Man5A, but a three-fold decrease in the production of Aa-Man5A. A preliminary investigation was performed to evaluate the possibility of using the recombinant Aa-Man5A in the processing of instant coffee. Arabica coffee extracts treated with Aa-Man5A displayed low viscosity in comparison to the untreated extract and showed better retention of volatile/aromatic compounds than the autoclaved extract. The results indicated that Aa-Man5A is capable of hydrolyzing coffee galactomannan and can be used for processing instant coffee. / AFRIKAANSE OPSOMMING: Mannaanpolisakkariede kom in die hemisellulose fraksie van plantselwande as strukturele polimere of reserwe koolstofbron voor. Mannaan word hoofsaaklik in die sade van peulplante, III die vorm van galaktomannaan, en III sagtehout as galaktoglucomannaan aangetref. Endo-I,4-j3-mannanase kan mannaanpolisakkariede na oligosakkariede van verskillende lengtes afbreek. Hierdie ensieme word deur fungi, bakterieë, plante en diere as enkele katalitiese modules of as deel van multi-modulêre proteïene uitgeskei. Die j3-mannanase (Aa-Man5A) van Aspergillus aculeatus is byvoorbeeld 'n enkele katalitiese module, maar die j3-mannanase (Tr-Man5A) van Trichoderma reesei bestaan uit 'n j3-mannanase katalitiese module gekoppel aan 'n sellulose-bindingsmodule deur middel van 'n Pro-Ser- Thr-ryke koppelstuk. Heteroloë geenuitdrukking in gIS bied die geleentheid om individuele hydrolitiese ensieme in 'n gasheer uitdrukkingsisteem sonder verwante aktiwiteite te produseer. Saccharomyces cerevisiae het 'n goed ontwikkelde uitdrukkingsisteem en word as model organisme vir heteroloë geenuitdrukking gebruik. 'n Aantal outoseleksiesisteme is ontwikkel, waardeur rekombinante S. cerevisiae-tese in enige medium sonder selektiewe druk gekweek kan word. 'n Outoseleksiesisteem, gebaseer op defektiewe chromosomale ura3 en furl gene wat vir ensieme in die pirimidien biosinteseweg kodeer, en komplementasie van die ura3-geen met die wilde-tipe URA3-geen wat op In multikopie plasmied teenwoordig is, is vir hierdie studie gebruik. Die manl-geen, wat vir die Aa-Man5A j3-mannanase van A. aculeatus kodeer, is gekloneer en in outoselektiewe S. cerevisiae onder die regulering van die alkoholdehidrogenase 2 (ADH2PT) en fosfogliseraatkinase 1 (POKl PT) promotor- en termineerderopeenvolgings uitgedruk. Uitdrukking van die manl-geen onder albei promotors het hoë produksievlakke van Aa-Man5A gelewer. Die produksievlakke was aansienlik hoër as die endo-I,4-j3-mannanase-vlakke wat deur heteroloë geenuitdrukking in Escherichia coli geproduseer was, en kon vergelyk word met die produksievlakke van ensieme in Pichia pastoris. P. pastoris is veronderstel om In hoër sekresiekapasiteit as S. cerevisiae te hê. Die rekombinante gisras wat die manl-geen onder beheer van die PGKl p promotor uitgedruk het, se biomassavorming was belemmer gedurende die laat logaritmiese fase. Die belemmering is opgehef nadat die natuurlike sekresiesein van 13-mannanasemet die MFais sekresiesein vervang is. Die rekombinante Aa-ManSA het soortgelyke biochemiese eienskappe as die natuurlike Aa-ManSA getoon. Die rekombinante ensiem het onvertakte mannaan tot hoofsaaklik mannose, mannobiose en mannotriose gehidroliseer. Die uitdrukking van die manl- en manl Licbd-geenkonstrukte van T. reesei in S. cerevisiae furl::LEU2-rasse onder regulering van die PGKlPT promotor en termineerder het tot die produksie en sekresie van onderskeidelik die Tr-ManSA en Tr-ManSAilCBD (sonder die sellulose-bindingsdomein) ensieme gelei. Die produksievlakke van beide proteïene was egter ongeveer IS-voudig laer as die vlakke van Aa-ManSA. Hierdie vlakke het egter nie verbeter nadat die natuurlike sekresiesein met die MFais sekresiesein vervang is nie. Interessant is die feit dat 'n afname in opkwekingstemperatuur vanaf 30°C tot 20°C tot 'n vyf-voudige toename m sekresievlakke van die Tr-ManSA gelei het, maar tot 'n drie-voudige afname in die produksie van Aa-ManSA. 'n Voorlopige ondersoek na die moontlike gebruik van rekombinante Aa-ManSA in kitskoffieprosessering is ondersoek.. Arabica koffie-ekstrak wat met Aa-ManSA behandel is, het 'n laer viskositeit in vergelyking met onbehandelde ekstrak getoon, asook beter behoud van vlugtige/aromatiese verbindings in vergelyking met geoutoklaveerde ekstrak. Hierdie resultate toon dat Aa-ManSA in staat is om koffee galaktomannaan te hidroliseer en dat dit vir die prosessering van kitskoffie gebruik kan word.

Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Saccharomyces cerevisiae uses the nitrogenous compounds in its environment selectively. The basis of this phenomenon is the transcriptional regulation of genes whose products are required for nitrogen catabolism. A rich nitrogen source represses the expression of genes required for the degradation of poor nitrogen sources via the action of the target of rapamyein (TOR) signaling cascade. If only a poor nitrogen source is available, these genes are derepressed. This process is known as nitrogen catabolite repression (NCR) or nitrogen regulation. The DALI and DAL4 genes of S. cerevisiae are transcribed divergently from the 829 bp intergenic region. The five known UASNTR elements (GATAI-5) were mutated in the full context of the intergenic promoter. All five elements are required for the transcriptional activation of DAL4. The two elements most proximal to DAL4 (GATA4 and GATA5) contributed the most and the one most distal (GATAI) contributed the least to its expression. In contrast, three of the five elements (GATA2-4) are required for DALI activation. In addition, analyses revealed that no single element is shared equally between these two genes. Predictions as to the function of known nitrogen-regulating elements based on their sequence and location proved to be inaccurate in some cases. Mutation analyses of the three UISALL elements present in the intergenic promoter region revealed that UIS8, which does not share a high degree of homology with the consensus UISALL sequence, is required the most for transcriptional induction of both DALI and DAL4. Also, UIS7, which shares the most similarity with the UISALL consensus sequence, has the phenotype of a repressor-like element when mutated. These observations therefore portray the opposite phenotypes of what was expected. We identified a regulator, Vid30p, which is required for the transcriptional response of S. cerevisiae in low ammonia conditions. Genetic analyses of the vid30/j, mutant indicate that Vid30p functions by regulating the expression of genes required for the production and degradation of glutamate. The transcription of VID30 is NCR-sensitive, highly induced by low concentrations of ammonia, and rapamycin-sensitive. In addition, the vid30/j, mutant is hypersensitive to rapamycin, indicating that this protein is, directly or indirectly, controlled by the TOR signaling pathway. / AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae het die vermoeë om stikstofbronne vanuit die omgewing selektief te benut. Die basis van hierdie verskynsel is die transkripsionele regulering van gene wat vir proteïene kodeer wat stikstof katabolisme bemiddel. 'n Goeie stikstofbron onderdruk die transkripsie van gene wat met die degradering van swak stikstofbronne gemoeid is. Hierdie onderdrukking word deur die teiken-van-rapamisien (TVR)-seintransduksiepad bewerkstellig. Wanneer slegs 'n swak stikstofbron beskikbaar is, word hierdie gene geaktiveer. Hierdie verskynsel staan as stikstofkatabolietonderdrukking (SKR) of stikstofregulering bekend. Die DALI- en DAL4-gene van S. cerevisiae word divergent vanaf 'n 829 bp intergeniese area getranskribeer. Vyf UASNTR-elemente (GATAI-5) is in die volle konteks van die intergeniese promotor gemuteer. Al vyf elemente word vir DAL4 transkripsionele aktivering benodig. Die twee elemente mees proksimaal tot DAL4 (GATA4 en GATA5) lewer die grootste bydrae tot DAL4-geenuitdrukking, terwyl die mees distale element (GATAI) die kleinste bydrae lewer. In teenstelling hiermee lewer slegs drie van die vyf elemente (GATA2-4) 'n noemenswaardige bydrae tot DALI se uitdrukking. Nie een van die vyf elemente lewer 'n gelykwaardige bydrae tot die uitdrukking van DALI en DAL4 nie. Voorspellings betreffende die bydrae van die onderskeie UASNTR-elemente tot die uitdrukking van die DALI- en DAL4-gene, gebaseer op die sekwens en die posisie van die element in die promotor, was meestal onakkuraat. Die drie U/SALL-elemente in die intergeniese area is gemuteer en toon dat U/S8, wat nie 'n groot mate van homologie met die U/SALL konsensus sekwens deel nie, die mees kritiese element vir transkripsionele induksie van beide DALI en DAL4 is. UIS7, wat 'n hoër mate van homologie met die UISALL konsensus sekwens deel, toon die fenotipe van 'n onderdrukkingselement wanner dit gemuteer word. Hierdie waarnemings is dus die teenoorgestelde van wat verwag is. Ons het 'n reguleerder, Vid30p, geïdentifiseer wat benodig word VIr die transkripsionele response van stikstofgereguleerde gene in lae konsentrasie ammonium. Genetiese analises van die vid3011 mutant toon dat Vid30p funksioneer deur die transkripsie van gene gemoeid met die vorming en degradering van glutamaat te reguleer. Die transkripsie van V/D30 is SKO-sensitief, word sterk deur lae konsentrasies ammonium geïnduseer, en is rapamisien-sensitief. Die vid30t!. mutant is ook hipersensitief vir rapamisien, wat aandui dat Vid30p, direk of indirek, deur die TVR-seintransduksiepad gereguleer word.

No description available.

No description available.

Expression of cDNA clones of the M1 double-stranded RNA killer preprotoxin coding region in Saccharomyces cerevisiae successfully directed the synthesis of secreted active toxin. Transformants harbouring these expression plasmids also displayed a K1 specific immunity phenotype. Immunoprecipitation of intracellular proteins with antitoxin antiserum showed that these transformants synthesize a 42kd glycosylated preprotoxin precursor. Two smaller unglycosylated immunoreactive species could also be resolved. These toxin precursor species were characterized by using secretory-defective hosts, by comparative electrophoretic mobilities, and by tunicamycin susceptibility. Such studies indicate that these protein species represent intermediates generated by signal cleavage of the preprotoxin and its subsequent glycosylation and provide evidence that these events occur post-translationally. Mutational analysis of the 44 amino acid preprotoxin N-terminal leader indicated that it is functionally bipartite, consisting of an N-terminal signal sequence and a C-terminal pro-sequence. Deletion of the leader perturbed but did not eliminate secretion of toxin.

No description available.

Reversible phosphorylation is a major mechanism for regulating the activity, localization and stability of proteins required for vital cellular processes such as glucose metabolism, gene expression, establishment of polarity, mitosis and cytokinesis. Phospho-regulation is driven by the activities of kinases and phosphatases. Together, these enzymes account for ∼3% of eukaryotic genomes and it is estimated that 30% of the eukaryotic proteome is composed of phospho-proteins. Protein kinases (PKs) have been studied extensively, however relatively little is known regarding the signaling networks of protein phosphatases (PPases). The identification of PPase functional networks has been slow due to the redundant nature of the majority of PPases, the complexity of their substrate recognition in vivo, and the lack of large-scale analyses that would facilitate network analysis. We hypothesized that large-scale analysis of genetic interactions using the Synthetic Genetic Array (SGA) and proteomic analyses using 2D-PAGE Difference Gel Electrophoresis (DiGE) could reveal PPase functional networks. Here, we apply this approach to the essential and conserved PP1 PPase Glc7 as it regulates numerous cellular processes in budding yeast. For this study, we created a glc7 hypomorphic mutant (glc7-E101Q) suited for both SGA and DiGE analyses. SGA analysis of glc7-E101Q revealed a broad network of 147 synthetic sick/lethal (SSL) and 178 synthetic rescue (SR) interactions. DiGE comparison of the glc7-E101Q proteome relative to wild-type at medium-resolution (∼1000 proteins) revealed alterations in 39 proteins that changed as a consequence of both the mutation and growth conditions. One of the proteins identified in this analysis was Eno1, a non-essential enolase that is mis-regulated in the presence of glucose and identified a SR mutation in the glc7-E101Q SGA. Subsequent phenotypic analysis suggests a novel, non-metabolic role for Eno1 in the Glc7 interaction network. Our results reveal that parallel analysis, using SGA and DIGE, can reveal novel functions and networks that a single analysis may not detect.

This thesis is a genetic study of the cytoplasmically- inherited determinant [psi] of Saccharomyces cerevisiae . [psi] is a potentiator of ochre suppression. The molecular basis of [psi] was investigated using mutagenesis as a probe. The psi⁺ phenotype (efficient suppression) can be mutated to psi^- phenotype (loss of suppression) by ultra-violet light (UV) and nitrosoguanidine (NTG) . The UV-induced mutation was a single-hit event and the pre-mutational lesion was partly photoreactivable . Repair or expression of UV-induced mutation to the [psi] determinant was under the same genetic control as for nuclear mutation. It was concluded that [psi] has a DNA genome. The 'extrachromosomal mutagens' thymidylate starvation, 5-fluorouracil, manganese chloride and cycloheximide failed to induce psi^- mutants whilst guanidine hydrochloride, dimethyl sulphoxide and potassium chloride were shown to induce this mutation at frequencies up to 100%. Several other physical and chemical agents caused a high frequency of loss of the psi⁺ phenotype. A new class of recessive nuclear mutation (pnm) was shown to cause a loss of the psi⁺ phenotype. A simple comple- mentation test was devised to distinguish them from cytoplasmic psi^- mutants. The dominant PNM^- mutation was shown not to cause a physical loss of the [psi] genome. Two mutants with a modified PNM^- phenotype were analysed. Attempts to demon- strate genetically the involvement of [psi] with the 80S ribosome were unsuccessful. The psi⁺ phenotype was conclusively demonstrated to be inherited independently' of the nucleus using a 'heterokaryon test'. Two models for the [psi] phenomena were proposed; one postulating the presence of a DMA 'plasmid' and one postulating the involvement of a stable, self-perpetuating metabolic state.

No description available.

No description available.