Indiana University-Purdue University Indianapolis (IUPUI) / Two important nutrient sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), participate in the control of yeast growth and metabolism in response to changes in nutrient availability. Starvation for amino acids activates the GAAC through Gcn2p phosphorylation of the translation initiation factor eIF2 and preferential translation of GCN4, a transcription activator. TOR senses nitrogen availability and regulates transcription factors, such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome during amino acid starvation and rapamycin treatment. We found that the GAAC is a major effector of the TOR pathway, with Gcn4p and Gln3p each inducing a similar number of genes during rapamycin treatment. While Gcn4p activates a common core of 57 genes, the GAAC directs significant variations in the transcriptome during different stresses. In addition to inducing amino acid biosynthetic genes, Gcn4p activates genes required for assimilation of secondary nitrogen sources, such as -amino-butyric acid (GABA). Gcn2p activation upon shifting to secondary nitrogen sources is suggested to occur by means of a dual mechanism. First, Gcn2p is induced by the release of TOR repression through a mechanism involving Sit4p protein phosphatase. Second, this eIF2 kinase is activated by select uncharged tRNAs, which were shown to accumulate during the shift to GABA medium. This study highlights the mechanisms by which the GAAC and TOR pathways are integrated to recognize changing nitrogen availability and direct the transcriptome for optimal growth adaptation.

Within the dissertation thesis “Study of Biological Material Attributes by Using Image Analysis Methods”, attention is focused on monitoring of the application of image analysis methods, mostly a fractal analysis, in studying the properties of various yeast species. The thesis includes determining the number of yeast cells and vegetative propagation of yeast using fractal parameters – fractal measure D and fractal dimension K. Attention is also paid not only to the application of the existing image analysis methods, but also to their renovation. The obtained images were evaluated using the box counting method specified by implementation of wavelet transformation. To monitor yeast cells for a longer time, it was first necessary to prepare a suitable microscopic preparation. To distinguish live and dead cells, the following fluorescent dyes were used: acridine orange, fluorescein diacetate, FUN-1, and Calcofluor White M2R. The images of yeast cells were recorded using a still camera or a CCD camera and microscope. Clips of the same size were obtained from the acquired digital photographs and processed by the HarFA program developed at the Faculty of Chemistry, Brno University of Technology. On the results it is possible to see a change in the fractal dimension depending on time, i.e. on the change of a budding cell structure, or to determine the number and radius of yeast cells upon predefined conditions.

Loss of heterozygosity (LOH) is considered a causal event in the formation of many cancers, with increasing evidence suggesting that DNA double-strand breaks (DSBs) play a major role in its occurrence. Despite its prominence in cancer, however, the precise molecular mechanisms responsible for extensive LOH and how such events are suppressed in normal cells is poorly understood. To investigate the mechanisms responsible for extensive break-induced LOH in eukaryotes, this study took advantage of an assay system in which such events could be identified through screening for loss of an auxotrophic his3⁺ marker, found ~25kb distal to an HO-endonuclease cut site in a non-essential minichromosome in Schizosaccharomyces pombe. Studies using this system had previously shown that extensive break-induced LOH in wild-type background, whilst infrequent, was predominantly associated with large translocations resulting from both allelic crossovers during G2 phase and breakinduced replication (BIR). Such extensive loss of allele specific information was also found to require rhp55⁺, rhp51⁺, rhp54⁺ and mus81⁺. This study has identified an additional role for the MRN complex, Rad22 and RPA in such break-induced translocations, suggesting that both allelic crossovers and BIR require homologous recombination (HR) in fission yeast. Surprisingly, break-induced extensive LOH was still observed in HR mutants. In contrast to wild-type cells, however, such extensive LOH was found to arise predominantly through de novo telomere addition at, or near, the break-site. Interestingly, telomere addition was most frequently observed in a rad22Δ background that disrupts HR following end resection. Further analysis demonstrated that de novo telomere addition was also significantly increased in ku70Δ rhp55Δ cells. Moreover, overexpression of rhp51 in rhp55Δ cells led to a substantial reduction in break-induced de novo telomere addition. Together, these findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ssDNA ends. In addition to providing information on break-induced LOH this study has identified a requirement for the MRN complex in efficient repair in rhp55Δ cells, which was previously found to occur via sister chromatid recombination (SCR) or a HRdependent end-joining pathway (EJ). Interestingly, deletion of MRN components also resulted in an increase in telomere addition, providing further evidence that HR competes with telomere addition for the repair of DSBs. Overall, these findings shed light on the competitive relationships between pathways of DSB repair/misrepair in S. pombe and how such mechanisms contribute to the prevention or promotion of genome instability.

Call number: LD2668 .T4 1979 W45 / Master of Science

No description available.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Basidiomycetous yeasts are found in a wide range of geographical areas ranging from tropical forests to desert regions. These yeasts are associated with different habitats such as soil, decaying vegetative debris, living plants and animals. Some may even be opportunistic human pathogens. In most of these habitats the yeasts may periodically be exposed to adverse conditions such as osmotic stress. Forty-one basidiomycetous yeast strains obtained from culture collections and isolated from nature were studied using various methods which includes the determination of different minimum water activities (aw; NaCl or sorbitol) for growth, survival in soil of varying moisture content, intracellular osmolytes accumulated and their release upon hypo-osmotic stress. The growth of most strains showed greater tolerance to NaCl than sorbitol at the same level of water activity. Interestingly, there were no basidiomycetous strains that showed growth below 0.90aw. 13C nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC) was used to analyze the osmolytes accumulated by all the strains of basidiomycetous yeasts when grown at 0.95 aw (NaCl). Glycerol was the major solute accumulated intracellulary by all the yeasts. Arabitol, mannitol or trehalose was accumulated in addition to glycerol in most yeasts whereas a number of yeasts only accumulated glycerol when grown at reduced aw. However, Cryptococcus laurentii US 1F was an exception by accumulating three solutes intracellularly when grown at reduced water activity. When exposed to hypo-osmotic shock all three solutes were rapidly released from the cells. Cryptococcus hungaricus CBS 5421, Cryptococcus macerans CBS 2206 and Cryptococcus neoformans US I1 were further analyzed with 13C NMR spectroscopy to determine whether the type of osmolyte accumulated during different phases of growth at 0.95aw (NaCl) might change. No changes were observed as the same osmolytes were accumulated in all cases. Five yeast strains (C. neoformans US I1, Rhodotorula mucillaginosa CBS 5951, C. macerans CBS 2206T, Filobasidium floriforme CBS 6240 and Sporidiobolus salmonicolor CBS 5937) were analyzed by HPLC for osmolytes released when exposed to hypo-osmotic shock. The strains differed in the pattern of response of osmolyte release. Only three strains released most of their osmolytes rapidly within 5 min, while C. macerans CBS 2206T and R. muculaginosa CBS 5951 retained most of the osmolytes intacellularly. This suggests that there might be different mechanisms of osmolyte release in basidiomycetous yeasts. A few strains of basidiomycetous yeasts (C. neoformans US I1, R. mucilaginosa CBS 5951, C. laurentii 1F, C. macerans CBS 2206T, F. floriforme CBS 6240, C. neoformans CBS 0132, C. laurentii CBS 0139, S. salmonicolor CBS 5937 and Filobasidium capsuligenum CBS 4381) were grown in soil cultures of different field capacity (100%, 25%, 10% and 5%) and evaluated for their survival in this environment. All the strains grew at 100% field capacity. Strains R. mucilaginosa CBS 5951, F. floriforme CBS 6240 and F. capsuligeum CBS 4381 also showed growth in soil at 25% field capacity. However, strains C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and C. laurentii CBS 0139 did not grow at this moisture content but survived up until the end of the experimental period. At lower soil moisture content (5% and 10% field capacity), the yeast strains either showed survival or decreased viability towards the end of the experimental period. Strain C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and R. mucilaginosa CBS 5951 and F. floriforme CBS 6240 showed survival at both 5% and 10% field capacity. However, strain F. neoformans CBS 0132, C. laurentii CBS 0139, F. capsuligenum CBS 4381 and S. salmonicolar CBS 5937 showed a decrease in viability after either 2 or 5 days of incubation. No relationship could be found between the type and number of intracellular osmolytes accumulated when exposed to osmotic stress (0.95aw NaCl) and the ability to grow and survive in soil with lower moisture content. Similarly, the ability of the yeasts to grow and survive in soil with lower moisture content did not correlate with their minimum aw for growth in a liquid medium. It was speculated that other factors, such as the physico-chemical composition of the soil, may also play a role in the survival of a particular yeast species in soil. This study has shown that the responses of basidiomycetous yeasts to reduced aw are physiologically similar to the ascomycetous yeasts. The types of osmolytes accumulated are similar but the basidiomycetous yeasts appear to be more sensitive to reduced aw and they tolerate NaCl better than sorbitol whereas the ascomycetous yeasts tolerate high sugar environments better. This is in agreement with the environments where these yeasts are usually found. / AFRIKAANSE OPSOMMING: Basidiomisete giste word aangetref in ‘n wye reeks geografiese areas, wat strek vanaf tropiese woude tot woestynstreke. Hierdie giste word geassosieer met verskillende habitatte soos grond, verrottende vegetatiewe reste, lewende plante en diere. Sommige mag selfs opportunistiese menslike patogene wees. By meeste van hierdie habitatte mag giste periodies blootgestel word aan moeilike toestande soos osmotiese stres. Een-en-veertig basidiomisete gisrasse, verkry vanaf kultuurversamelings en geisoleer vanuit die natuur, was bestudeer met verskeie metodes, waaronder die bepaling van verskillende minimum water aktiwiteite (aw; NaCl of sorbitol) vir groei, droë massa bepalings, akkumulasie van intrasellulêre osmoliete, asook hul vrystelling met hipo-osmotiese stres. Meeste rasse het meer weerstand teen NaCl as sorbitol gehad by dieselfde vlak van wateraktiwiteit. Dit was interessant om op te let dat geen basidiomisete stamme groei onder 0.90aw getoon het nie. Beide 13C kern magnetiese resonansie (KMR) spektroskopie en hoë uitset vloeistof chromatografie (HUVC) was gebruik om alle opgehoopde osmoliete te analiseer vir alle basidiomisete gisrasse tydens groei tot by 0.95 aw (NaCl). Vir alle giste was gliserol die opgelosde stof wat die meeste intrasellulêr opgehoop het. Arabitol of mannitol of trehalose het saam met gliserol in meeste giste opgehoop, terwyl ‘n aantal giste slegs gliserol opgehoop het tydens groei by verlaagde aw. Cryptococcus laurentii US 1F was daarenteen ‘n uitsondering deurdat dit drie opgelosde stowwe intrasellulêr versamel het tydens groei by verlaagde wateraktiwiteit. Al drie hierdie opgelosde stowwe is uit die selle vrygesel na blootstelling aan hipo-osmotiese skok. Verdere 13C KMR spektroskopie analise was gedoen op Cryptococcus hungaricus CBS 5421, Cryptococcus macerans CBS 2206 en Cryptococcus neoformans US I1 om vas te stel of die tipe opgehoopde osmoliet tydens verskillende fases van groei by 0.95aw (NaCl) mag verander. Geen veranderinge was egter waargeneem aangesien dieselfde osmoliete in alle gevalle opgehoop het. Vyf gisrasse (C. neoformans US I1, Rhodotorula mucillaginosa CBS 5951, C. macerans CBS 2206T, Filobasidium floriforme CBS 6240 en Sporidiobolus salmonicolor CBS 5937) was geanaliseer deur HUVC vir osmolietvrystelling tydens blootstelling aan hipo-osmotiese skok. Die rasse het verskil in hul responspatroon van osmolietvrystelling. Slegs drie rasse het meeste van hul osmoliete vrygestel binne die eerste 5 minute, terwyl ander, C. macerans CBS 2206T en R. muculaginosa CBS 5951 meeste van hul osmoliete intrasellulêr teruggehou het. Dit dui daarop dat daar dalk verskillende meganismes van osmolietvrystelling in basidiomisete giste mag voorkom. Etlike basidiomisete gisrasse (C. neoformans US I1, R. mucilaginosa CBS 5951, C. laurentii 1F, C. macerans CBS 2206T, F floriforme CBS 6240, C. neoformans CBS 0132, C. laurentii CBS 0139, (B) S. salmonicolor CBS 5937, Filobasidium capsuligenum CBS 4381) was opgegroei in grondkulture van verskillende veldkapasiteit (100%, 25%, 10% en 5%) en ondersoek vir hul oorlewing in hierdie omgewing. Al die rasse kon groei by ‘n 100% veldkapasiteit. Die rasse R. mucilaginosa CBS 5951, F. floriforme CBS 6240 en F. capsuligeum CBS 4381 kon ook groei in grond met 25% veldkapasiteit. Alhoewel rasse C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T en C laurentii CBS 0139 nie kon groei by hierdie voginhoud nie, het hulle nog steeds oorleef tot aan die einde van die eksperimentele tydperk. By verlaagde grond voginhoud (5% en 10% veldkapasiteit) het die gisrasse of oorleef of ‘n verlaagde lewensvatbaarheid openbaar teen die einde van die eksperimentele prosedure. Die rasse C. neoformans US I1, C. laurentii US 1F, C. macerans CBS 2206T and R. mucilaginosa CBS 5951 en F. floriforme CBS 6240 het oorleef by beide 5% en 10% veldkapasiteit. Die rasse F. neoformans CBS 0132, C. laurentii CBS 0139, F. capsuligenum CBS 4381 en S. salmonicolar CBS 5937 het egter ‘n verlaging in lewensvatbaarheid getoon na ‘n periode van of 2 of 5 dae inkubasie. Geen ooreenstemming kon gevind word tussen die tipe en aantal intrasellulêre opgehoopde osmoliete nie na blootstelling aan osmotiese druk (0.95aw NaCl), asook hul vermoë om te groei en oorleef in grond met ‘n laer voginhoud. Terselfdertyd het die giste se vermoë om te groei en oorleef in grond met ‘n laer voginhoud nie ooreengestem met hul minimum aw vir groei in vloeibare groeimedium nie. Ter spekulasie kan ander faktore, soos die fisio-chemiese samestelling van die grond ook ‘n rol speel in die oorlewing van ‘n spesifieke gisspesie in grond. Hierdie studie het getoon dat basidiomisete giste se respons tot verlaagde aw fisiologies dieselfde is as vir askomisete giste. Alhoewel die tipe versamelde osmoliete identies was, wil dit voorkom asof basidiomisete giste meer sensitief teenoor ‘n verlaagde aw is, tesame met ‘n beter bestandheid teen NaCl as sorbitol, terwyl askomisete giste hoë suiker omgewings beter kan verdra. Dit is in ooreenstemming met die tipe omgewings waar hierdie giste gewoonlik voorkom.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and involve the interplay of a multitude of proteins. A unique collection of thermosensitive secretory mutants allowed scientists to demonstrate that the secretory pathway of the yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All proteins commence their journey in the endoplasmic reticulum, where they undergo amino-linked core glycosyl modification. After passage through the Golgi apparatus, where the remodelling of the glycosyl chains is completed, proteins are transported to their final destinations, which are either the cell surface, periplasmic space or the vacuole. Proteins destined for secretion are usually synthesised with a transient amino-terminal secretion leader of varying length and hydrophobicity, which plays a crucial role in the targeting and translocation of their protein cargo. Considerable effort has been made to elucidate the molecular mechanisms involved in these processes, especially due to their relevance in a rapidly expanding biotech industry. The advantages of S. cerevisiae as a host for the expression of recombinant proteins are well documented. Unfortunately, S. cerevisiae is also subject to a number of drawbacks, with a relative low product yield being one of the major disadvantages. Bearing this in mind, different secretion leaders were compared with the aim of improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for its ability to degrade raw starch and an improved secretion of its amylase enzymes from S. cerevisiae paves the way for a potential one-step starch utilisation process. Three sets of constructs were prepared containing the LKA 1 and LKA2 genes separately under secretory direction of either their native secretion leader, the S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1 secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p proteolytic processing of the leader/protein fusion. Strains expressing the amylase genes under their native secretion leaders resulted in the highest saccharolytic activity in the culture medium. In contrast to this, strains utilising the synthetic secretion leader produced the highest fermentation yield, but had a lower than expected extracellular activity. We hypothesise that the native amylase leaders may function as intramolecular chaperones in the folding and processing of their passenger proteins, thereby increasing processing efficiency and concomitant enzyme activity. / AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat gestelom die ooreenkoms tussen die sekresiepad van die gis Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur die Golgi-apparaat beweeg het, waar die laaste veranderings aan die glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die seloppervlak, die periplasmiese ruimte of die vakuool, vervoer. Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike, amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf. Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die relatiewe lae produkopbrengs die belangrikste is. Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van 'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Wine is the product of a complex biological and biochemical interaction between grapes and different microorganisms (fungi, yeasts, lactic acid bacteria and acetic acid bacteria, as well as the mycoviruses and bacteriophages affecting them) in which yeasts play the most important role regarding the alcoholic (primary) fermentation. These wine-associated yeasts can be divided into Saccharomyces and non-Saccharomyces yeasts. During fermentation, there is a sequence of dominance by the various non-Saccharomyces yeasts, followed by Saccharomyces cerevisiae, which then completes the fermentation. This is especially evident in spontaneously fermenting must, which has a low initial S. cerevisiae concentration. Some non- Saccharomyces yeasts can also be found throughout the fermentation. The non- Saccharomyces presence in the fermentation can affect wine quality, either positively or negatively. A positive contribution could be especially useful to improve wines produced from grape varieties with a neutral flavour profile due to non-optimal climatic conditions and/or soil types. As part of a comprehensive South African research programme, the specific objectives of this study were: the isolation of indigenous non-Saccharomyces yeasts from vineyards and musts; the identification of these isolates; the characterisation and evaluation of predominant species under winemaking conditions; and the development of a protocol for their use in enhancing wine quality. Initially, 720 isolates representing 24 different species, were isolated from grape (vineyard) and must samples taken over three vintages from four distinctly different wine producing regions. The isolates were characterised and grouped utilising biochemical profiles and DNA karyotyping, whereupon representative isolates were identified. The yeast species that had the highest incidence of predominance in the vineyard was Kloeckera apiculafa. However, some vineyard samples were characterised by low numbers or absence of this yeast, which is not according to generally accepted norms. Other species that also predominated in a few of the vineyard samples were Candida pulcherrima, Kluyveromyces thermofolerans, Rhodotorula sp. and Zygosaccharomyces bailii. Generally, there was a greater diversity of yeasts in the processed must than from the vineyard samples. Furthermore, while each sample showed a different yeast population, no pattern linking species to climatic zone was observed. Four species i.e. Candida collieulosa, Candida pulcherrima, Candida stel/ata and Kloeckera apiculata, were found to predominate in grape must samples. Representative strains consequently received further attention during laboratory and small-scale winemaking trials. A protocol was developed whereby individual species could be used in co-inoculated fermentations with S. cerevisiae in the small-scale production of wine. An improvement in wine quality was achieved and it was found that there was a link between specific species and grape cultivar. The ability of C. pulcherrima to improve Chenin blanc wine quality was investigated further. Results over three vintages showed that the wine produced by the co-inoculated fermentation was superior to that of a reference wine (produced by S. cerevisiae only). The improvement in wine quality was not linked to increased ester content nor were the standard chemical analyses adversely affected. The effects of pH and wine production parameters i.e. 802, fermentation temperature and use of di-ammonium phosphate (DAP), on this yeast followed the same pattern as that known for S. cerevisiae. This study was successfully completed and the developed protocol can be used for the improvement of Chenin blanc wine where additional aroma and quality is needed. / AFRIKAANSE OPSOMMING: Wyn is die produk van 'n komplekse biologiese en biochemiese interaksie tussen druiwe en mikroorganismes (swamme, giste, melksuurbakterieë, asynsuurbakterieë, asook die mikovirusse en bakteriofage wat hul beïnvloed) waar gis die belangrikste rol speel ten opsigte van die alkoholiese (primêre) fermentasie. Die betrokke giste kan in Saccharomyces- en nie-Saccharomyces-giste verdeel word. Tydens gisting vind daar 'n opeenvolging van dominansie deur die verskillende nie-Saccharomyces giste plaas, gevolg deur Saccharomyces cerevisiae, wat dan die gisting voltooi. Dit is veral in spontaan fermenterende mos, waarin aanvanklik lae konsentrasies S. cerevisiae-gisselle voorkom, waarneembaar. Sekere nie-Saccharomyces-giste kan ook regdeur die verloop van fermentasie gevind word. Die teenwoordigheid van nie-Saccharomyces-giste kan 'n bydrae maak tot wynkwaliteit, hetsy positief of negatief. 'n Positiewe bydrae kan veral nuttig wees vir die verbetering van wyn geproduseer van druifsoorte met neutrale geurprofiele as gevolg van nie-optimale klimaatstoestande en/of grondtipes. As deel van 'n uitgebreide Suid-Afrikaanse navorsingsprogram, was die doelwitte van hierdie studie soos volg: die isolasie van inheemse nie-Saccharomyces-giste vanuit wingerde en mos; die identifikasie van hierdie isolate; die karakterisering en evaluering van spesies wat tydens wynbereiding oorheers; en die ontwikkeling van 'n protokol waarin geselekteerde nie- Saccharomyces-giste gebruik kan word vir die verbetering van wynkwaliteit. Druif- en mosmonsters is oor drie oestye vanuit vier duidelik onderskeibare wynproduserende gebiede geneem en 720 isolate, verteenwoordigend van 24 verskillende spesies, is hieruit geïsoleer. Hierdie isolate is volgens biochemiese profiele en DNA-kariotipering gekarakteriseer en gegroepeer waarna verteenwoordigende isolate geïdentifiseer is. Die gisspesie wat die meeste in wingerde voorgekom het, was Kloeckera apiculata. Sommige wingerde is egter deur lae getalle of afwesigheid van dié gis gekenmerk, In feit wat afwyk van die algemeen aanvaarde norm. Ander spesies, nl. Candida pulcherrima, Kluyveromyces thermotolerans, Rhodotorula sp. en Zygosaccharomyces bailii, het ook in enkele gevalle in die wingerdmonsters oorheers. Oor die algemeen was daar 'n groter diversiteit van giste in die geprosesseerde mos as in die wingerdmonsters. Verder is elke monster gekenmerk deur verskillende gispopulasies, maar geen verband tussen gisspesie en klimaatsone is waargeneem nie. Vier spesies, nl. Candida collieulosa, Candida pulcherrima, Candida stel/ata en Kloeckera apiculata, het in hoë getalle in die druiwemosmonsters oorheers en verteenwoordigende rasse het verdere aandag tydens laboratorium- en kleinskaalse wynmaakproewe geniet. 'n Protokol, waar hierdie rasse individueel gebruik is in gesamentlike geïnokuleerde fermentasies met S. cerevisiae vir die kleinskaalse produksie van wyn, is ontwikkel. 'n Verbetering in wynkwaliteit is verkry en daar is 'n verband tussen spesifieke gisspesies en druifvariëteit gevind. Gevolglik is die vermoë van C. pulcherrima om die gehalte van Chenin blanc wyn te verbeter, verder ondersoek. Resultate oor drie oesjare het gewys dat die wyn wat met die C. pulcherrima / S. cerevisiae kombinasie geproduseer is, beter was as 'n verwysingswyn (deur slegs S. cerevisiae geproduseer). Die waargenome verbetering in wynkwaliteit was egter nie aan 'n verhoging in esterinhoud te danke nie en die standaard chemiese analises het geen negatiewe afwyking uitgewys nie. Verder is gevind dat die effek van pH en wynproduksieparameters, nl. die gebruik van S02, fermentasietemperatuur en die gebruik van di-ammoniumfosfaat (DAP), dieselfde patroon as die bekend vir S. cerevisiae gevolg het. Die ontwikkelde protokol kan nou aangewend word waar verhoogde Chen in blanc wynaroma en kwaliteit verlang word.

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: In the yeast Saccharomyces cerevtstee, two biochemical pathways ensure that activated cytoplasmic or peroxisomal acetyl-groups are made available for mitochondrial energy production when the cells utilise non-fermentable carbon sources. The first pathway is the glyoxylate cycle, where two activated acetyl-groups are incorporated into each cycle, which releases a C4 intermediate. This intermediate is then transported to the mitochondria where it can enter the tricarboxylic acid cycle. The second pathway is the carnitine shuttle. Activated acetyl-groups react with carnitine to form acetylcarnitine, which is then transported to the mitochondria where the acetyl group is transferred. In this study it was shown that the deletion of the glyoxylate cycle specific citrate synthase, encoded by CIT2, results in a strain that is dependent on carnitine for growth on non-fermentable carbon sources. Using a /::"cit2 strain, mutants affected in carnitine-dependent metabolic activities were generated. Complementation of the mutants with a genomic library resulted in the identification of four genes involved in the carnitine shuttle. These include: (i) the mitochondrial and peroxisomal carnitine acetyltransferase, encoded by CAT2; (ii) the outer-mitochondrial carnitine acetyltransferase, encoded by YA T1; (iii) the mitochondrial carnitine translocase, encoded by CRC1; and (iv) a newly identified carnitine acetyltransferase, encoded by YAT2. All three carnitine acetyltransferases are essential in a carnitine-dependent strain. The dependence on exogenous carnitine of the /::"cit2 strain when grown on nonfermentable carbon sources suggested that S. cerevisiae does not biosynthesise carnitine. Measurements using electrospray mass spectrometry confirmed this hypothesis. As a result an investigation was initiated into carnitine biosynthesis in order to genetically engineer a S. cerevisiae strain that could endogenously biosynthesise carnitine. The filamentous fungus, Neurospora crassa, was one of the first organisms used in the seventies to identify the precursor and intermediates of carnitine biosynthesis. However, it was only about twenty years later that the first genes encoding these enzymes where characterised. Carnitine biosynthesis is a four-step process, which starts with trimethyllysine as precursor. Trimethyllysine is converted to hydroxytrimethyllysine by the enzyme trimethyllysine hydroxylase (TMLH). Hydroxytrimethyllysine is cleaved to trimethylamino-butyraldehyde by the hydroxytrimethyllysine aldolase (HTMLA) releasing glycine. Trimethylaminobutyraldehyde is dehydrogenated to trimethylamino-butyrate (y-butyrobetaine) by trimethylamino-butyraldehyde dehydrogenase (TMABA-DH). In the last step, ybutyrobetaine is converted to t-carnltine by y-butyrobetaine hydroxylase (BBH). The N. crassa TMLH homologue was identified in the genome database based on the protein sequence homology of the human TMLH. Due to the high amount of introns predicted for this gene, the cDNA was cloned and subjected to sequencing, which then revealed that the gene indeed had seven introns. Functional expression of the gene in S. cerevisiae and subsequent enzymatic analysis revealed that the gene coded for a TMLH. It was therefore named cbs-1 for "carnitine biosynthesis gene no. 1JJ. Most of the kinetic parameters were similar to that of the human TMLH enzyme. Following this, a genomic copy of the N. crassa BBH homologue was cloned and functionally expressed in S. cerevisiae. Biochemical analysis revealed that the BBH enzyme could biosynthesise L-carnitine from y-butyrobetaine and the gene was named cbs-2. In addition, the gene could rescue the growth defect of the carnitinedependent Scii? strain on non-fermentable carbon sources when y-butyrobetaine was present. This is the first report of an endogenously carnitine biosynthesising strain of S. cerevisiae. The cloning of the remaining two biosynthesis genes presents particular challenges. To date, the HTMLA has not been characterised on the molecular level making the homology-based identification of this protein in N. crassa impossible. Although the TMABA-DH has been characterised molecularly, the protein sequence is conserved for its function as a dehydrogenase and not conserved for its function in carnitine biosynthesis, as in the case of TMLH and BBH. The reason for this is probably due to the fact that the enzyme is involved in other metabolic processes. The use of N. crassa carnitine biosynthesis mutants would probably be one way in which to overcome these obstacles. The !1cit2 mutant proved useful in studying carnitine related metabolism. We therefore searched for suppressors of !1cit2, which resulted in the cloning of RAS2. In S. cerevisiae, two genes encode Ras proteins, RAS1 and RAS2. GTP-bound Ras proteins activate adenylate cyclase, Cyr1 p, which results in elevated cAMP levels. The cAMP molecules bind to the regulatory subunit of the cAMP-dependent kinase (PKA), Bcy1 p, thereby releasing the catalytic subunits Tpk1 p, Tpk2p and Tpk3p. The catalytic subunits phosphorylate a variety of regulators and enzymes involved in metabolism. Overexpression of RAS2 could suppress the growth defect of the Sclt? mutant on glycerol. In general, overexpression of RAS2 enhanced the proliferation of wild-type cells grown on glycerol. However, the enhancement of proliferation was much better for the !1cit2 strain grown on glycerol. In this respect, the retrograde response may play a role. Overexpression of RAS2 resulted in elevated levels of intracellular citrate and citrate synthase activity. It therefore appears that the suppression of !1cit2 by RAS2 overexpression is a result of the general upregulation of the respiratory capacity and possible leakage of citrate and/or citrate synthase from the mitochondria. The phenotype of RAS2 overexpression contrasts with the hyperactive RAS2val19 allele, which causes a growth defect on glycerol. However, both RAS2 overexpression and RAS2val19activate the cAMP/PKA pathway, but the RAS2val19dependent activation is more severe. Finally, this study implicated the Ras/cAMP/PKA pathway in the proliferation effect on glycerol by showing that in a Mpk1 strain, the growth effect is blocked. However, the enhanced proliferation was still observed in the Mpk2 and Mpk3 strains when RAS2 was overexpressed. Therefore, it seems that Tpk1 p plays an important role in growth on non-fermentable carbon sources, a notion that is supported by the literature. / AFRIKAANSE OPSOMMING: In die gis Saccharomyces cerevtstee, is daar twee metaboliese weë waarmee geaktiveerde asetielgroepe na die mitochondrium vervoer kan word wanneer die sel op nie-fermenteerbare koolstofbronne groei. Die een weg is die glioksilaatsiklus, waar die geaktiveerde asetielgroepe geïnkorporeer word in die siklus en dan vrygestel word as Ca-intermediêre. Hierdie intermediêre word dan na die mitochondrium vervoer waar dit in die trikarboksielsuursiklus geïnkorporeer word. Die ander weg is die karnitiensiklus, waar geaktiveerde asetielgroepe met karnitien reageer om asetielkarnitien te vorm wat dan na die mitochondrium vervoer word waar dit die asetielgroep weer vrygestel. Hierdie studie het getoon dat die delesie van die glioksilaatsiklus spesifieke sitraatsintetase, gekodeer deur CIT2, die gisras afhanklik maak van karnitien vir groei op nie-fermenteerbare koolstofbronne. Deur gebruik te maak van 'n ócit2 gisras, kon mutante, wat geaffekteer is in karnitien-verwante metaboliese aktiwiteite, gegenereer word. Komplementering van die mutante met 'n genomiese biblioteek het gelei tot die identifisering van vier gene betrokke by die karnitiensiklus. Hierdie gene sluit in: (i) die mitochondriale en die peroksisomale karnitienasetieltransferase, gekodeer deur CAT2; (ii) die buite-mitochondriale karnitienasetieltransferase, gekodeer deur YAT1; (iii) die mitochondriale karnitientranslokase, gekodeer deur CRC1; en (iv) 'n nuutgeïdentifiseerde karnitienasetieltransferase, gekodeer deur YAT2. Daar benewens, is ook gewys dat al drie karnitienasetieltransferases noodsaaklik is in 'n karriltienafhanklike gisras. Die afhanklikheid van eksogene karnitien van die ócit2 gisras, wanneer dit gegroei word op nie-fermenteerbare koolstofbronne, was aanduidend dat S. cerevisiae nie karnitien kan biosintetiseer nie. Metings deur middel van elektronsproeimassaspektrometrie het hierdie veronderstelling bevestig. Gevolglik is 'n ondersoek deur ons geïnisieer in die veld van karnitienbiosintese om 'n S. cerevisiae gisras geneties te manipuleer om karnitien sodoende endogenies te biosintetiseer. Die filamentagtige fungus, Neurospora crassa, was een van die eerste organismes wat in die sewentiger jare gebruik is om die voorloper en intermediêre van karnitienbiosintese te identifiseer. Dit was egter eers sowat twintig jaar later dat die eerste gene wat vir hierdie ensieme kodeer, gekarakteriseer is. Karnitienbiosintese is 'n vierstap-proses wat met trirnetlellisten as voorloper begin. Trimetiellisien word omgeskakel na hidroksi-trimetiellisien deur die ensiem trimetiellisienhidroksilase (TMLH). Hidroksietrimetlelllsien word dan gesplits om trimetielaminobuteraldehied te vorm deur die werking van die hidroksitrimetiellisienaldolase (HTMLA) met die gevolglike vrystelling van glisien. Trimetielaminobuteraldehied word dan na trimetielaminobuteraat (y-butirobeteïen) deur trimetielaminobuteraldehied dehidrogenase (TMABA-DH) gedehidrogeneer. In die laaste stap word y-butirobeteïen deur middel van die y-butirobeteïen hidroksilase (BBH) na L-karnitien omgeskakel. Op grond van die proteïenvolgordehomologie in die genoomdatabasis tussen die menslike TMLH en N. crassa se TMLH is laasgenoemde geïdentifiseer. As gevolg van die groot getal introns wat vir hierdie geen voorspel is, is die cDNA-weergawe daarvan gekloneer en aan volgordebepaling onderwerp. Dit het getoon dat die geen inderdaad sewe introns bevat. Funksionele uitdrukking van die geen in S. cerevisiae en ensiematiese analise het getoon dat die geen vir 'n TMLH kodeer en is gevolglik cbs-1 genoem; dit staan vir "karnitien biosintese geen no. 1tt. Meeste van die kinetiese parameters was ook soortgelyk aan die van die menslike TMLH-ensiem. Hierna is 'n genomiese kopie van N. crassa se BBH-homoloog gekloneer en funksioneel in S. cerevisiae uitgedruk. Biochemiese analise het getoon dat die uitgedrukte BBH-ensiem L-karnitien vanaf y-butirobeteïen kan biosintetiseer en die geen is cbs-2 genoem. Daar benewens kon die geen die groeidefek van die karnitien-afhanklike tlcit2-gisras ophef wanneer dit op nie-fermenteerbare koolstofbronne in die teenwoordigheid van y-butirobeteïen aangekweek is. Hierdie is die eerste verslag oor 'n endogeniese karnitien-biosintetiserende ras van S. cerevisiae. Die klonering van die oorblywende twee karnitienbiosintetiserende gene het sekere uitdagings. Tot op datum, is die HTMLA nog nie tot op genetiese vlak gekarakteriseer nie, wat dan die homologie-gebaseerde identifikasie van hierdie proteïen in N. crassa onmoontlik maak. Alhoewel die TMABA-DH geneties gekarakteriseer is, is die proteïenvolgorde ten opsigte van sy funksie as 'n dehidrogenase gekonserveer, maar nie vir sy funksie in karnitienbiosintese soos in die geval van TMLH en BBH nie. Die rede hiervoor is moontlik omdat die ensiem ook in ander metaboliese prosesse betrokke is. Die gebruik van N. crassa karnitienmutante sal moontlik een manier wees om hierdie probleme te oorkom. Die tlcit2-mutant het handig te pas gekom vir die bestudering van karnitienverwante metabolisme. Dus is daar vir onderdrukkers van die tlcit2-mutant gesoek wat gelei het tot die klonering van die RAS2-geen. In S. cere visiae , kodeer twee gene vir Ras-proteïene, RAS1 en RAS2. GTP-gebonde Ras-proteïene aktiveer adenilaatsiklase, Cyr1 p, wat verhoogde intrasellulêre cAMP-vlakke tot gevolg het. Die cAMP bind aan die regulatoriese subeenheid van die cAMP-proteïenkinase (PKA), Bcy1 p, en daardeur word die katalitiese subeenhede, Tpk1 p, Tpk2p en Tpk3p, vrygestel. Die katalitiese subeenheid fosforileer 'n verskeidenheid van reguleerders en ensieme betrokke by metabolisme. Ooruitdrukking van RAS2 het die groeidefek van die tlcit2-mutant op gliserolonderdruk. Oor die algemeen, verbeter die ooruitdrukking van RAS2 die proliferasie van die wildetipe op gliserol bevattende media. Alhoewel, die verbetering van proliferasie was baie meer opmerklik in die tlcit2-gisras. In hierdie verband, speel die gedegenereerde response dalk 'n rol. Ooruitdrukking van RAS2 het verhoogde intrasellulêre vlakke van sitraat- en sitraatsintetase-aktiwiteit tot gevolg gehad. Dit wou dus voorkom asof die onderdrukking van die ócit2-groeidefek deur RAS2 se ooruitdrukking die gevolg was van algemene opreguiering van respiratoriese kapasiteit en die lekkasie van sitraat en/of sitraatsintetase uit die mitochondria. Die fenotipe van RAS2 ooruitdrukking kontrasteer die hiperaktiewe RAS2va / 19 alleel, wat 'n groeidefek op gliserol media veroorsaak. Alhoewel beide RAS2-00ruitdrukking en RAS2va / 19 die cAMP/PKA-weg aktiveer, is gevind dat die RAS2va/19-afhanklike aktivering strenger is. Ten slotte, die cAMP/PKA-weg is in die proliferasie effek op gliserol media geïmpliseer deur te wys dat in 'n Mpk1-gisras, die groeieffek geblokkeer is. Alhoewel, die verbeterde proliferasie is steeds waargeneem in die Mpk2-en Mpk3-gisrasse toe die RAS2-geen ooruitgedruk is. Dus, dit wil voorkom asof Tpk1 p 'n belangrike rol in die groei van gisselle op nie-fermenteerbare koolstofbronne speel; 'n veronderstelling wat deur die literatuur ondersteun word.

Dissertation (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study: 1. Compares various heterologous expression systems for high-level expression of cytochromes P450. Limitations of the existing cytochromes P450 expression systems are discussed and possibilities to improve the expression yields of human steroidogenic enzymes, are suggested. In addition the potential applications of human steroidogenic cytochromes P450 expressed in Pichia pastoris are illustrated. 2. Describes the cloning and extracellular expression of a recombinant full-length human cytochrome P450 17a-hydroxylase (p45017a) enzyme in Saccharomyces cerevisiae. After the optimisation of expression conditions, it was shown that this system is not suitable for the expression of full-length human P45017a. 3. Describes the cloning and extracellular expression of the full-length human cytochromes P45017a, aromatase, bs and truncated human cytochrome P45017a in P. pastoris. The limitations using P. pastoris as an export system for expressed P450 enzymes were pointed out. 4. Describes the cloning and intracellular expression of the full-length human cytochrome P45017a in P. pastoris as well as the functional expression of human P45017a in P. pastoris, showing progesterone conversion to 17ahydroxyprogesterone and 16a-hydroxyprogesterone in vivo, for the first time. 5. Evaluates developed methods for the preparation of mierosomes from P. pastoris expressing human P45017a and the spectral characterisation of detergent solubilised human P45017a. 6. Describes the development of protocols for the purification of human P45017a from P. pastoris microsomes. / AFRIKAANSE OPSOMMING: Hierdie ondersoek: 1. Vergelyk verskillende heterologiese proteïen uitdrukkings-sisteme vir die preperatiewe produksie van sitochrome P450. Die tekortkomings van bestaande sitochroom P450-uitdrukkings-sisteme word bespreek en moontlikhede om die opbrengs van menslike steroïedogeniese ensieme te verbeter word voorgestel. Die potensiële aanwendings van menslike steroïedogeniese sitochrome P450, wat in Pichia pas/oris uitgedruk word, word ook geïllustreer. 2. Beskryf die klonering en ekstrasellulêre uitdrukking van die rekombinante vollengte menslike sitochroom P45017a-hidroksilase (P45017a) ensiem in Saccharomyces cerevisiae. Na optimisering van die kondisies vir die uitdrukking kon aangetoon word dat hierdie sisteem nie geskik is vir die uitdruk en sekresie van vollengte menslike P45017a nie. 3. Beskryf die klonering en ekstrasellulêre uitdrukking van die vollengte menslike sitochrome P45017a, aromatase, b5 en verkorte menslike sitochroom P45017a in P. pas/oris. Die beperkinge van P. pas/oris as 'n uitvoersisteem vir die uitdrukking van P450 ensieme word bespreek. 4. Besryf die klonering en intrasellulêre ekspressie van die vollengte menslike sitochroom P450 17a. Die funksionele ekspressie van menslike sitochroom P450 17a in P. pas/oris is vir die eerste keer gekarakteriseer. 5. Evalueer die ontwikkelde metodes vir die voorbereiding van mikrosome van P. pas/oris wat menslike P45017a uitdruk en karakteriseer die detergent opgelosbare menslike P45017a t.o.v. spektroskopiese eienskappe. 6. Beskryf die ontwikkelling van protokolle vir die suiwering van die uitgedrukte menslike P45017a vanuit P. pas/oris mikrosome.