Thesis (PhD)--University of Stellenbosch, 2002 / ENGLISH ABSTRACT: Upon nutrient limitation, normal cells of the budding yeast, Saccharomyces cerevisiae, undergo a transition from ovoid cells that bud in an axial (haploid) or bipolar (diploid) fashion to elongated cells that bud in a unipolar fashion. The daughter cells stay attached to the mother cells, resulting in chains of cells referred to as pseudohyphae. These filaments can grow invasively into the growth substrate (haploid), or away from the colony (diploid), and are hypothesised to be an adaptation of yeast cells that enables them to search for nutrientrich substrates. This filamentous growth response to nutrient limitation was shown to be dependent on the expression of, amongst others, the MUC1 gene. MUC1 (also known as FL011) encodes a large, cell wall-associated, GPI-anchored threonine/serine-rich protein that bears structural resemblance to mammalian mucins and to the yeast flocculins. Deletion and overexpression studies demonstrated that it is critical for pseudohyphal differentiation and invasive growth, and that overexpression of the gene also results in strongly flocculating yeast strains. The upstream regulatory region of MUC1 comprises the largest yeast promoter identified to date and areas as far as 2.4 kb upstream of the translational start site have been shown to confer regulation on MUC1 expression. The large promoter region is not unique to MUC1, however, since it is almost identical to that of the functionally unrelated STA2 gene. The STA2 gene, as well as the identical STA1 and STA3 genes, encodes extracellular glucoamylase isozymes that enable the yeast cell to utilise starch as a carbon source. Glucoamylases liberate glucose residues from the non-reducing end of the starch molecule, thereby making it accessible to yeast cells. The high identity between the promoters of MUC1 and STA1-3 suggests that the two genes are co-regulated. In addition, several transcription factors that regulate the transcriptional levels of both MUC1 and STA2 have been identified and include Msn1p and the previously uncharacterised Mss11p. Overexpression of either Msn1p or Mss11p results in elevated levels of MUC1 and STA2 transcription and a dramatic increase in flocculation, invasive growth, pseudohyphal differentiation and the ability to utilise starch, suggesting that the two genes are indeed co-regulated. The main objective of this study was to characterise Mss11p and its role in the co-regulation of MUC1 and STA2 (as a representative member of the STA gene family). A detailed expression analysis, using Northern blots and Lacl reporter gene expression studies in different media, confirmed that these genes are indeed co-regulated to a large extent. MUC1 and STA2 are also regulated by the same transcriptional regulators, which include not only Msn1pand Mss11p, but also Ste12p, the transcription factor of the mating pheromone/filamentous growth signalling cascade, and Flo8p, a transcriptional activator of the flocculation genes. Overexpression of the genes encoding these factors results in elevated expression levels of both MUC1 and STA2 in most nutritional conditions and enhances the filamentous growth phenotypes of the strain, as well as the ability to degrade starch. On the other hand, the deletion thereof results in severe reductions in the transcription levels of MUC1 and STA2, with equally severe reductions in filamentous growth and the ability to hydrolyse starch. These expression studies also showed that the repressive effect of STA10, a previously uncharacterised negative regulator of STA2, is actually a phenotype conferred by a FLOB mutation in some laboratory strains of S. cerevisiae. The upstream regulatory regions of MUC1 and STA2 are the largest promoters in the yeast genome. By sequencing the upstream areas of STA2 and STA3 and comparing them to the sequence of MUC 1, it was shown that these upstream areas are 99.7%identical over more than 3 900 base pairs (bp) upstream of the translational start. With the exception of a few minor substitutions, the only significant difference between the MUC1 and STA2 promoters is the presence of a 20-bp and a 64-bp sequence found in the MUC1 promoter, but not in the promoters of any of the STA1-3 genes. Through a promoter-deletion analysis, it was shown that Mss11p, Msn1pand Flo8p exert their control over the transcription of MUC1 and STA2 from an 90-bp sequence located at -1 160 to -1 070 in the STA2 and -1 210 to -1 130 in the MUC1 promoters. This sequence also mediates the effect of carbon catabolite repression on the transcription of STA2 and MUC1. Despite the similarities in the expression patterns of MUC1 and STA2, some discrepancies also exist. The most significant difference is that, in wild-type cells and under all nutritional conditions tested, MUC1 transcription is reduced significantly if compared to the transcription levels of STA2. This was attributed to the presence of the 20- and 64-bp sequences, that are present in the promoter region of MUC1, but absent from that of STA2. To place the transcriptional regulators of MUC1 and STA2 in the context of known signal transduction pathways, an epistasis analysis was conducted between MSN1, MSS11 and components of the mating pheromone/filamentous response MAPkinase cascade and cAMPPKA pathway that were shown to be required for the filamentous growth response. This analysis revealed that Msn1p functions in a third, as yet uncharacterised, signal transduction pathway, also downstream of Ras2p,but independent of the two identified pathways, i.e. the cAMP-PKA and pheromone response/filamentous growth response MAP kinase pathways. However, Mss11p seems to function downstream of all three the identified pathways. This suggestsa critical and central role for Mss11p in determining the transcription levels of MUC1 and STA2. To further characterise Mss11p and its role in the transcriptional regulation of MUC1 and STA2, it was also subjected to a detailed deletion and mutation analysis. Mss11p was shown to harbour two distinct activation domains required for the activation of MUC1 and STA2, but also able to activate a reporter gene expressed from under the GALl promoter. The more prominent of the activation domains of Mss11p was shown to be one of the domains with homology to Flo8p, designated H2. The H2 domain has significant homology to a number of proteins of unknown function from a range of different organisms. A multi-sequence alignment allowed the identification of conserved amino acids in this domain. Mutations in two of the four conserved amino acid pairs in the H2 domain completely eliminated the activation function of Mss11p. The poly-glutamine and poly-asparagine domains of Mss11p are not required for its activation function. The deletion of these domains has no impact on the ability of Mss11p to activate MUC1 or STA2 or of the Gal4p-Mss11p fusion to activate the lacl reporter gene expressed from under the GAL7 promoter. Gal4p fusions of either of these domains were also unable to trans-activate the PGAL7-lacl reporter gene. As such, it was concluded that neither of these domains performs a function in the role of Mss11p as a transcriptional activator. We also demonstrated that the putative ATP/GTP-binding domain (P-loop) is not required for the transcriptional activation function of Mss11p. In an attempt to identify other target genes of Mss11p, the use of micro-arrays was employed to assessthe impact of the overexpression and deletion of MSS11 on the total yeast transcriptome. These results showed that MUC1 and STA2 are the only two genes in the ISP15 genetic background that are significantly (more than 15-fold) enhanced by the overexpression of MSS11. Mss11p therefore seemsto playa very specific or dedicated role in MUC1 and STA2 transcription. This analysis also identified several genes (DBP2, ROM2, YPLOBOC, YGR053C, YNL179C, YGR066C) that are repressed by overexpression of MSS11 and activated when MSS11 is deleted. To integrate all the results, three possible models for the activation of MUC1 and STA2 transcription by Mss11p are proposed: (i) Mss11p performs the role of a transcriptional mediator, possibly in a protein complex, to convey information from upstream regulatory elements to the transcription machinery assembledat the core promoters of MUC1 and STA2; (ii) Mss11p plays a more direct role in transcriptional activation, possibly as a transcription factor itself; and (iii) Mss11p facilitates transcription of the MUC1 and STA2 promoters as part of a larger complex that removes or releases the chromatin barrier over the MUC1 and STA2 promoters in responseto specific nutritional signals. / AFRIKAANSE OPSOMMING: Wanneer voedingstowwe beperkend raak, ondergaan selle van die botselvormende gis, Saccharomyces cerevisiae, fn transformasie vanaf ronde selle, wat in fn aksiale (haploïede) of bipolêre (diploïede) patroon bot, tot verlengde selle, wat slegs op een punt bot. Die dogterselle blyaan die moederselle geheg, sodat kettings van selle, wat as pseudohifes bekend staan, gevorm word. Hierdie filamente kan fn groeisubstraat binnedring (haploïede) of vanaf die kolonie weggroei (diptoïede), en is moontlik fn aanpassing van die gisselle wat hulle in staat stelom na meer voedingstofryke substrate te groei. Die vermoë om filamente in respons tot voedingstoftekorte te vorm, is onderhewig aan die uitdrukking van, onder meer, die MUC1-geen. MUC1 (ook bekend as FL011) kodeer vir fn selwand-geassosieerde treonien/serien-ryke proteten met fn GPI-anker wat strukturele verwantskappe met die mukiene van soogdiere en die flokkuliene van giste toon. Delesie- en ooruitdrukkingstudies het bewys dat dit krities is vir die ontwikkeling van pseudohifes en penetrerende groei, terwyl die ooruitdrukking daarvan ook tot sterk flokkulerende gisrasse lei. Die stroom-op regulatoriese area van MUC1 vorm die grootste promotor wat tot dusver in gis geïdentifiseer is, en daar is bewys dat areas so ver as 2.4 kb stroom-op van die translasie-inisiëringsetel die regulering van MUC1 beïnvloed. Hierdie groot promotor is egter nie uniek tot MUC1 nie, aangesien fn amper identiese promotor die regulering van die funksioneelonverwante STA2-geen beheer. Die STA2-geen, asook die identiese STA1- en STA3-gene, kodeer vir ekstrasellulêre glukoamilase isosieme wat die gis in staat stelom stysel as koolstofbron te benut. Dit bevry glukosemolekules vanaf die nie-reduserende punt van die styselmolekuul en stel dit sodoende aan gisselle beskikbaar. Die hoë vlak van eendersheid tussen dié twee promotors veronderstel dat die twee gene op soortgelyke wyse gereguleer word. Verskeie transkripsiefaktore wat die transkripsievlakke van beide MUC1 en STA2 beheer, is ook geïdentifiseer, Dit sluit Msn1p en die tot dusver ongekarakteriseerde Mss11p in. Ooruitdrukking van Msn1p of Mss11p lei tot verhoogde vlakke van MUC1 en STA2 se transkripsie en fn dramatiese toename in flokkulasie, asook die vermoë om penetrerend te groei, pseudohifes te vorm en stysel te benut. Dit bevestig dat die twee gene wel tot fn groot mate op dieselfde wyse gereguleer word. Die hoofdoel van hierdie studie was om Mss11p en die rol daarvan in die regulering van MUC1 en STA2 te karakteriseer. Gedetailleerde uitdrukkingsanalises met behulp van die Northern-kladtegniek en facZverklikkergeeneksperimente in verskillende media het bevestig dat die gene wel tot fn groot mate op dieselfde wyse gereguleer word. Transkripsie van MUC1 en STA2 word ook deur dieselfde transkripsionele reguleerders beheer, wat nie net Msn1pen Mss11p insluit nie, maar ook Ste12p, die transkripsiefaktor van die paringsferomoon/filamentagtige groei seintransduksiekaskade, en Fl08p, fn transkripsionele aktiveerder van die flokkulasiegene. Ooruitdrukking van die gene wat vir hierdie faktore kodeer, veroorsaak verhoogde uitdrukkingsvlakke van beide MUC1 en STA2 onder die meeste groeitoestande en verbeter die vermoë van die gisras om filamentagtig te groei en om stysel te benut. Andersyds veroorsaak delesies van die gene 'n dramatiese afname in die transkripsievlakke van MUC1 en STA2, met vergelykbare afnames in die vermoë van die gisras om filamentagtig te groei en om stysel te benut. Hierdie uitdrukkingstudies het ook bewys dat die onderdrukkingseffek van STA10, 'n tot dusver ongekarakteriseerde, negatiewe reguleerder van STA2, aan 'n mutasie in FLOB in sekere laboratoriumrasse van S. cerevisiae toegeskryf kan word. Die stroom-op regulatoriese areas van MUC1 en STA2 is die grootste promotors in die gis se genoom. Deur die nukleotiedvolgordes van die ver stroom-op areas van STA2 en STA3 te bepaal en hulle met dié van MUC1 te vergelyk, is daar vasgestel dat die stroom-op areas van die gene 99.7% identies is oor meer as 3 900 basispare (bp) stroom-op van die beginsetel van translasie. Met die uitsondering van enkele basispaarverskille, is die enigste noemenswaardige verskil tussen die promotors van MUC1 en STA2 die teenwoordigheid van 'n 20 bp- en 'n 64 hp-fragment wat in die MUC1-promotor aangetref word, maar nie in die promotors van die STA1-3 gene nie. Deur 'n promotordelesie-analise kon daar bewys word dat Mss11p, Msn1p en Flo8p beheer uitoefen oor die transkripsie van MUC1 en STA2 vanaf 'n 90-bp-fragment, wat by posisie -1 160 tot -1 070 in die STA2-promotor en posisie -1 210 tot -1 130 in die MUC1-promotor aangetref word. Koolstofkatabolietonderdrukking van MUC1 en STA2 se transkripsie geskied ook deur middel van hierdie fragment. Ten spyte van die ooreenkomste in die uitdrukkingspatrone van MUC1 en STA2, kom daar tog ook verskille voor. Die mees opvallende verskil is dat, in wilde-tipe selle en onder alle toestande tot dusver getoets, die transkripsievlakke van MUC1 aansienlik laer is as dié van STA2. Dit word toegeskryf aan die teenwoordigheid van die 20 bp- en 64 bp-fragmente, wat in die promotor van MUC1 teenwoordig is, maar in die promotor van STA2 afwesig is. Om die transkripsionele reguleerders van MUC1 en STA2 in die konteks van bekende seintransduksieweë te plaas, is 'n epistase-analise gedoen tussen MSN1, MSS11 en komponente van die paringsferomoon/filamentagtige groei MAP-kinasekaskade en die cAMPPKA- weg wat uitgewys het dat dit 'n rol in die filamentagtige groeirespons speel. Hierdie analise het onthul dat Msn1p in 'n derde, tot dusver onbeskryfde, seintransduksieweg funksioneer, wat ook stroom-af van Ras2p is, maar wat onafhanklik funksioneer van die twee bekende weë, die cAMP-PKA-weg en die paringsferomoon/filamentagtige groei MAPkinasekaskade. Mss11p blyk egter stroom-af van al drie dié weë te funksioneer. Dit wys dat Mss11p 'n kritiese en sentrale rol in die bepaling van MUC1 en STA2 se transkripsievlakke speel. Om Mss11p en die rol daarvan in die regulering van MUC1 en STA2 se transkripsie verder te karakteriseer, is dit aan 'n volledige delesie- en mutasie-analise onderwerp. Dit het gewys dat Mss11p twee verskillende aktiveringsdomeine bevat wat vir die transkripsionele aktivering van STA2 en MUC1 benodig word, maar wat ook 'n verklikkergeen kon aktiveer wat onder die GAL7-promotor uitgedruk word. Die prominentste van die twee aktiveringsdomeine van Mss11p is een van die domeine wat homologie toon met 'n soortgelyke domein van Flo8p, die sogenaamde H2-domein. Die H2-domein toon hornologie met 'n verskeidenheid van organismesse proteïene, waarvan die funksie onbekend is. 'n Vergelyking van al die relevante aminosuurvolgordes uit dié proteïene het gehelp om 'n aantal gekonserveerde aminosure te identifiseer. Mutasies van twee van die vier gekonserveerde aminosuurpare het die vermoë van Mss11p om transkripsie te aktiveer, heeltemal geëlimineer. Die poliglutamien- en poliasparagiendomeine van Mss11p word nie vir die aktiveringsfunksie benodig nie. Die delesie van die domeine het geen impak gehad op die vermoë van Mss11p om die transkripsie van MUC1 en STA2 te aktiveer nie, of op die vermoë van die Gal4p-Mss11p fusie om die lacZ-verklikkergeen onder regulering van die GAL7-promotor te aktiveer nie. Gal4p-fusies met enige van die domeine was ook nie in staat om die PGAL7-lacZverklikkergeen te aktiveer nie. Daar kan dus afgelei word dat nie een van die twee domeine 'n funksie in die rol van Mss11p as transkripsionele aktiveerder het nie. Soortgelyke eksperimente het bewys dat die moontlike ATP/GTP-bindingsdomein (P-lus) nie vir die transkripsionele aktiveringsfunksie van Mss11p benodig word nie. In 'n poging om ander teikengene van Mss11p te identifiseer, is mikro-ekspressieroosters gebruik om die impak van die ooruitdrukking en delesie van MSS11 op die totale transkriptoom van die gis te bepaal. Dié resultate het gewys dat MUC1 en STA2 die enigste twee gene in die ISP15genetiese agtergrond is waarvan transkripsie noemenswaardig (meer as 15-voudig) deur die ooruitdrukking van MSS11 verhoog word. Dit wil dus voorkom asof Mss11p 'n baie spesifieke rol in die transkripsie van MUC1 en STA2 speel. Hierdie analise het ook verskeie gene (DBP2, ROM2, YPLOBOC,YGR053C, YNL179C, YGR066C) geïdentifiseer wat deur die ooruitdrukking van MSS11 onderdruk word en deur die delesie van MSS11 geaktiveer word. Ten einde al die resultate te integreer, word drie moontlike modelle vir die aktivering van MUC1- en STA2-transkripsie deur Mss11p voorgestel: (i) Mss11p vervul die rol van 'n transkripsionele tussenganger, moontlik as deel van 'n proteïenkompleks, om die inligting van die stroom-op regulatoriese elemente aan die transkripsiemasjinerie wat oor die kernpromotor van MUC1 en STA2 gebind is, oor te dra; (ii) Mss11p speel 'n meer direkte rol in transkripsionele aktivering, moontlik as 'n transkripsiefaktor self; en (iii) Mss11p maak die transkripsie van MUC1 en STA2 moontlik as deel van 'n groter kompleks wat die chromatienblokkade oor die promotors van STA2 en MUC1 in respons tot spesifieke seine verslap of verwyder.

No description available.

Isoleucyl-tRNA synthetase genes from the yeast Saccharomyces cerevisiae and the ciliated protozoan Tetrahymena thermophila were sequenced. The intronless S. cerevisiae gene (ILS1) encodes a putative polypeptide of 1072 amino acids. Two putative promoter elements were identified, one for general amino acid control and one for constitutive transcription. A heat shock protein gene lies upstream of ILS1. The T. thermophila isoleucyl-tRNA synthetase gene (ilsA: formerly cupC) has eight introns, four transcription start sites, and codes for a putative polypeptide of 1081 amino acids with two leucine-zippers. These eukaryotic isoleucyl-tRNA synthetases are 47% identical. They are compared to homologous enzymes from Escherichia coli and an archaebacterium, and to other aminoacyl-tRNA synthetases. / Intron sequences and junctions from T. thermophila and other eukaryotes were analyzed and all but yeast and mammalian introns were found to be A + T enriched. T. thermophila transcription start sites were analyzed and occur at a T or an A within the consensus sequence (A/T)$ sb{ rm n}$ T A A (A)$ sb{ rm n}.$

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No description available.

Directed evolution is a powerful tool that mimics the natural selection process to engineer biomolecules with improved and altered functionalities for a wide variety of applications. The advance of biological engineering based on directed evolution techniques depends upon selection assays that can practically search large, diverse libraries for the most improved variants. In Chapter 1, we begin by discussing the potential of growth selections to serve as accessible and robust assays for directed evolution. We then delve into the existing approaches to expand the scope of targets for growth selections beyond those that are intrinsically linked to growth and the strategies implemented to improve their throughput and sensitivity. The yeast three-hybrid (Y3H) assay is a versatile system that can expand the field of directed evolution if implemented as a growth selection for the search of large variant libraries. Although the Y3H assay has been successfully applied as a positive selection to evolve proteins with improved functions, its expansion into applications requiring a high-throughput, versatile selection against transcriptional activation has been hindered by its limited dynamic range as a counter selection. To address the limited dynamic range of the Y3H assay, we undertook a multi-pronged approach to reengineer our Y3H counter selection to have a high dynamic range. In Chapter 2, we discuss strategies to improve the dynamic range of the Y3H counter selection by maximizing the growth between cells with activated and basal reporter gene expression levels. Specifically, we elaborate on the development and characterization of two Y3H counter selections that were based on either reporter gene degradation or an alternative phototoxic reporter gene. In Chapter 3, we present our most successful strategy to improve the dynamic range of the Y3H counter selection that uses the dual tetracycline (Tet) system to increase transcriptional regulation of the reporter gene. We employed a guided strategy based on both rational design and library approaches to find the best Tet Y3H reporter gene construct with the highest dynamic range. We believe our method to engineer the best Tet Y3H reporter construct will be widely useful to synthetic biologists developing sophisticated in vivo assays that require fine-tuned reporter gene expression levels. Finally, in Chapter 4, we demonstrate the versatility of the Y3H system by developing a screen for the detection of natural product biosynthesis. This assay should have an impact for metabolic engineers that are employing directed evolution techniques to generate large metabolic pathway libraries for the overproduction of high-value small molecules in heterologous producer strains.

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"September, 2002." / Bibliography: leaves 93-106. / 106 leaves : ill. (some col.), plates ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / In this thesis the biochemical and genetic characterization of Fkh2p identifies it as a major component of SFF. It has been shown to bind DNA in an Mcm1p dependent manner and the Fkh2p DNA binding domain is essential for this interaction. The protein interaction domain of Mcm1p has been demonstrated to be essential for ternary complex formation. Fkh2p, along with a functionally redundant protein Fkh1p, has been show to control the periodic expression of the CLB2 cluster genes. The functional characterisation of the Fkh2p domains reveals an important role for both the Forkhead associated domain and the C-terminus. Ndd1p. another protein important for mitotic progression, is shown to be important for CLB2 cluster regulation by de-repressing Fkh2p and activating gene expression. The role of cdk activity is shown to act through the CLB2 cluster upstream activating sequences, possibly through Ndd1p. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2003

In budding yeast, many of the genes that encode enzymes required for DNA precursor synthesis (MCB genes) are expressed under cell cycle control in late G1/S. The relationship between MCB gene expression, dNTP synthesis and DNA synthesis was investigated by using �� factor-synchronized Saccharomyces cerevisiae. The levels of all four dNTPs increased several-fold when cells crossed the G1/S boundary. An even larger increase in the dNTP pools occurred at G1/S when replication initiation was blocked by incubating synchronized dbf4 mutants at the nonpermissive temperature. Thus, dNTP accumulation at G1/S was not dependent on replication initiation. Similarly, MCB gene induction at G1/S was also independent of replication initiation. The accumulation of dNTPs at G1/S was dependent on Swi6, a protein known to be required for normal MCB gene regulation during the cell cycle. Treatment with hydroxyurea, an inhibitor of ribonucleotide reductase, blocked DNA synthesis and prevented the increase in dNTP levels that normally occurred at G1/S, however, it did not exhaust the basal levels of any of the four dNTPs. The mechanism responsible for replication arrest despite the persistence of dNTPs was not dependent on the checkpoint protein Rad53, as rad53 mutants also failed to exhaust basal dNTPs when incubated in HU. The inhibitory effect of HU on DNA synthesis was bypassed when dbf4 cells were allowed to pre-accumulate dNTPs at 37��C before being released to the permissive temperature in the presence of HU. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, as dbf4 cells incubated in HU at 25��C were able to initiate replication when cells were switched to the nonpermissive temperature and HU was removed. The results indicate that DNA chain elongation in yeast requires a critical dNTP threshold, below which replication forks are completely arrested. Cells lacking a functional thioredoxin system were deficient in dNTP synthesis. The rate of accumulation was significantly lower in ��trr1 mutants lacking thioredoxin reductase, and dNTP accumulation at G1/S did not occur at all in ��trxl ��trx2 double mutants lacking thioredoxin. The results suggest that thioredoxin serves as the electron donor for ribonucleotide reductase during DNA precursor synthesis in yeast. / Graduation date: 2003