No description available.

No description available.

Herpes simplex virus protein VP16 activates immediate-early (IE) viral gene expression upon infection. VP16-mediated transactivation depends on formation of a multi protein complex with cellular factors on a cis-acting TAATGARA T sequence present in the IE promoters. The potent acidic activation domain, contained within the carboxyl terminus of VP16, is dispensable for the complex formation. The amino terminal part of VP16, which is inert in transactivation in mammalian cells, is sufficient for selective interactions with cellular factors, one of which has been identified as the ubiquitous transcription factor Oct-1. The yeast two-hybrid system was utilized to isolate the cellular factor(s) necessary in addition to Oct-1 for VP16 induced complex formation. This system, designed to directly clone proteins interacting with a given protein of interest, employs the yeast transcriptional activator GAL4. An interaction between VP16 and the cellular factor(s), fused to GAL4 DNA binding and activation domain, respectively, reconstitutes a hybrid transactivator that stimulates expression of a reporter lacZ gene in yeast. Thus, (beta)-galactosidase activity serves as a positive signal for protein-protein interaction. As a prelude of using this method for isolation of VP16-interacting cellular proteins, the system was tested with HSV-1 protein vhs, known to bind to VP16 in vitro. The obtained data demonstrated an interaction between VP16 and vhs in the two-hybrid system and deletion analysis revealed that VP16 sequence contained within the first 369 amino acids is required for binding to vhs. Thus, VP16 residues necessary for interaction with vhs in vivo coincide with these identified previously for VP16-vhs complex formation in vitro. VP16 fused to the GAL4 DNA binding domain activated expression of the reporter lacZ gene in yeast, despite the absence of its acidic activation domain. Deletion analysis showed that the amino terminal 369 residues of VP16 were sufficient for transactivation in yeast. Similar GAL4-VP16 derivatives were inactive in mammalian cells as measured by transient transfection assays. Thus, unlike in yeast, VP16 lacking the acidic activation domain is deficient in transactivation in mammalian cells even if it is directly bound to a promoter. VP16 sequences required for complex formation with vhs overlaps with those implicated in interaction with the mammalian factors, indicating that this region is involved in protein-protein interactions with both cellular and viral factors. Consistent with this, VP16 interaction with a yeast factor supplying an activation domain in trans would explain VP16-dependent transactivation in the absence of its acidic activation domain. Alternatively, a yeast specific activation domain might be present in the amino terminal part of VP16. / Thesis / Master of Science (MS)

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.

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

The three-dimensional spatial architecture of chromosomes is integrally connected to chromatin function. Budding yeast telomeres cluster at the nuclear periphery, the ribosomal genes are localised to the nucleolus, tRNA genes may also tend to localise to the nucleolus or centromeres, while the later cluster near the spindle pole body. Recently, in the fission yeast Schizosaccharomyces pombe, a novel role has been revealed for the RNA polymerase III transcriptional apparatus, and TFIIIC in particular, in chromosome spatial organisation and boundary function. In this project, I investigate whether Saccharomyces cerevisiae Extra TFIIIC (ETC) sites, which bind the TFIIIC transcription factor but do not recruit RNA polymerase III, act to position chromosomal domains. I show that six of the eight known S. cerevisiae ETC sites localise predominantly at the nuclear periphery. An ETC site retains its tethering function when moved to a new chromosomal location. TFIIIC binding is necessary for peripheral localisation, since deleting the TFIIIC binding consensus ablates ETC site peripheral positioning. I find that any of the six TFIIIC subunits can drive peripheral tethering, suggesting that the TFIIIC complex is central to the positioning mechanism. Interestingly, anchoring of ETC sites to the nuclear periphery also requires Mps3, a Sad1-UNC-84 domain protein that spans the inner nuclear membrane. Moreover, I show that the mechanism of ETC site peripheral tethering requires chromatin remodelling proteins, and in particular Histone 3 - Lysine 56 (H3K56) acetylation. Finally, I investigate the biological function of ETC sites and examine the connection between this biological function and their ability to anchor at the nuclear periphery. In summary, TFIIIC and Mps3 together position a new class of genomic loci crucial for correct spatial organisation of S. cerevisiae chromosomes.

The yeast Saccharomyces cerevisiae decodes CAG codons using tRNAGlnCUG, encoded by the single-copy gene SUP70. On rich medium, the sup70-65 and sup70-33, alleles induce pseudohyphal growth, ordinarily a response to nitrogen limitation. sup70-65 tRNA is additionally a UAG stop codon suppressor. Characterisation of sup70 diploids revealed that they form pseudohyphal-like structures on both rich and N-deprived liquid, but not solid medium. Unlike canonical pseudohyphae, which bud in a unipolar fashion and are RAS2Val19 stimulated, sup70 pseudohyphal cells budded in a bipolar fashion that was RAS2Val19–resistant. Site-directed mutants of sup70-65 and sup70-33 that restored base complementarity in the mutant tRNA stems partially complimented the pseudohyphal phenotype and revealed that structural rigidity, rather than sequence identity, was the key phenotypic driver. A library of sup70 mutants, screened for novel nonsense UAG suppressor alleles, indentified several novel sites in the anticodon stem that induce tRNA distortion and UAG recognition. None of the new alleles induced formation of pseudohyphae. The sup70-65 pseudohyphal phenotype was weakly complemented through overexpression of the isoacceptor tRNAGlnUUG, which can inefficiently decode CAG via 3rd base wobble pairing, inferring pseudohyphal growth can be triggered by inefficient translation of CAG codons. Supporting this observation, tRNA Northern blotting revealed that all pseudohyphae-inducing sup70 mutants have reduced overall levels of tRNAGlnCUG, as well as reduced levels of tRNAGlnCUG charging. A systemic comparison of wild-type and sup70-65 proteomes using stable isotope labelling with amino acids revealed that 28 proteins showed significant changes in expression in the mutant, including 4 with known roles in control of bud size and/or budding pattern. This study revealed that pseudohyphae-inducing sup70 mutations compromise tRNAGlnCUG structure and amino acid charging and thus slow translation of cognate CAG codons, probably down-regulating the expression of a yet-to-be identified gene affecting the control of pseudohyphal differentiation.

Com o aumento de pacientes imunocomprometidos nas últimas décadas, os fungos têm emergido como uma das causas de doenças humanas. Leveduras ubíquas e/ou comensais como Saccharomyces cerevisiae, Rhodotorula sp., Kodamaea (Pichia), Trichosporon sp. estão sendo descritas como importantes causadoras de infecções. Com o objetivo de avaliar clinicamente os casos de infecções por leveduras emergentes, foi realizado um estudo de 101 casos diagnosticados da Irmandade Santa Casa de Misericórdia de Porto Alegre nos últimos anos, revisando a apresentação clínica, condição predisponente, terapia utilizada e evolução dos pacientes. A doença de base mais frequente foi insuficiência renal. A manifestação clínica principal foi febre. Os fatores de risco mais frequentes foram uso de cateter venoso central e internação em unidade de terapia intensiva. O antifúngico mais utilizado no tratamento das infecções por S. cerevisiae, Kodamaea ohmeri e Trichosporon sp. foi o fluconazol e no tratamento das infecções por Rhodotorula sp. foi a anfotericina B. Aproximadamente 32% dos pacientes apresentaram melhora clínica após o tratamento com antifúngico e 26% foram a óbito. O diagnóstico rápido e específico destas leveduras é importante para a decisão terapêutica e o melhor prognóstico. / With the increase of immunocompromised patients in recent decades, fungi have emerged as a major cause of human diseases. Ubiquitous yeast and/or commensal as Saccharomyces cerevisiae, Rhodotorula sp., Kodamaea (Pichia), Trichosporon sp. are described as important causative agents of infections. Faced with this, the identification of yeast is important for therapeutic decisions and for epidemiological studies. In order to clinically evaluate the cases of yeast infections emerging, a study of the major cases diagnosed at Irmandade Santa Casa de Misericordia de Porto Alegre in recent years, reviewing the clinical presentation, predisposing condition, therapy and patients’s progress. The underlying disease frequently was renal failure. The major clinical manifestation was fever. The most frequent risk factors were: use of central venous catheter and intensive care unit stay. The most used antifungal in the treatment of infections caused by S. cerevisiae, Kodamaea ohmeri and Trichosporon sp. was fluconazole and in the cases of infections by Rhodotorula sp. was amphotericin B. Most patients showed clinical improvement after treatment with antifungal. Approximately 32% of patients showed clinical improvement after treatment with antifungal and 26% died.The rapid and specific diagnosis of these yeasts is important for the therapeutic decision and a better prognosis.

No description available.