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Systems biology studies the complex interactions between components of biological systems. One major goal of systems biology is to reconstruct the network of interactions between genes in response to normal and perturbed conditions. In order to accomplish this goal, large-scale data are needed. Accordingly, diverse powerful and high-throughput methods must be developed for this purpose. We have developed novel high-throughput technologies focusing on cellular phenotype profiling and now provide additional genome-scale analysis of gene and protein function. Few high-throughput methods can perform large-scale and high-throughput cellular phenotype profiling. However, analyzing gene expression patterns and protein behaviors in their cellular context will provide insights into important aspects of gene function. To complement current genomic approaches, we developed two technologies, the spotted cell microarray (cell chip) and the yeast spheroplast microarray, which allow high-throughput and highly-parallel cellular phenotype profiling including cell morphology and protein localization. These methods are based on printing collections of cells, combined with automated high-throughput microscopy, allowing systematic cellular phenotypic characterization. We used spotted cell microarrays to identify 15 new genes involved in the response of yeast to mating pheromone, 80 proteins associated with shmoo-tip 'localizome' upon pheromone stimulation and 5 genes involved in regulating the localization pattern of a group II intron encoded reverse transcriptase, LtrA, in Escherichia coli. Furthermore, in addition to morphology assays, yeast spheroplast microarrays were built for high-throughput immunofluorescence microscopy, allowing large-scale protein and RNA localization studies. In order to identify additional cell cycle genes, especially those difficult to identify in loss-of-function studies, we performed a genome-scale screen to identify yeast genes with overexpression-induced defects in cell cycle progression. After measuring the fraction of cells in G1 and G2/M phases of the cell cycle via high-throughput flow cytometry for each of ~5,800 ORFs and performing the validation and secondary assays, we observed that overexpression of 108 genes leads to reproducible and significant delay in the G1 or G2/M phase. Of 108 genes, 82 are newly implicated in the cell cycle and are likely to affect cell cycle progression via a gain-of-function mechanism. The G2/M category consists of 87 genes that showed dramatic enrichment in the regulation of mitotic cell cycle and related biological processes. YPR015C and SHE1 in the G2/M category were further characterized for their roles in cell cycle progression. We found that the G2/M delay caused by the overexpression of YPR015C and SHE1 likely results from the malfunction of spindle and chromosome segregation, which was supported by the observations of highly elevated population of large-budded cells in the pre-M phase, super-sensitivity to nocodazole, and high chromosome loss rates in these two overexpression strains. While the genes in the G2/M category were strongly enriched for cell cycle associated functions, no pathway was significantly enriched in the G1 category that is composed of 21 genes. However, the strongest enrichment for the G1 category consists of the genes involved in negative regulation of transcription. For instance, the overexpression of SKO1, a transcription repressor, resulted in strong cell cycle delay at G1 phase. Moreover, we found that the overexpression of SKO1 results in cell morphology changes that resembles mating yeast cells (shmoos) and activates the mating pheromone response pathway, thus explaining the G1 cell cycle arrest phenotype of SKO1 ORF strains.

Hybridization studies based on the prototrophic selection technique, involving the use of auxotrophic mutants of strains of all accepted species of the genus Kluyveromyces, are reported. Two main groups of mutually interfertile taxa were established within the genus. The first group comprises Kluyveromyces bulgaricus, Kluyveromyces cicerisporus, Kluyveromyces dobzhanskii, Kluyveromyces drosophilarum, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces phaseolosporus, Kluyveromyces vanudenii and Kluyveromyces wikenii. The second group consists of Kluyveromyces dabzhanskii, Kluyveromyces drosophilarum, Kluyveromyces laotis, Kluyveromyces vanudenii and Kluyveromyces wiokerhamii. Hybrids were also detected in crosses involving Kluyveromyces drosophilarum and Kluyveromyces waltii as well as Kluyveromyces marxianus and Kluyveromyces thermotolerans. In terms of the concept of the biological species and in compliance with the requirements of the International Code of Botanical Nomenclature, taxa which hybridize with Kluyveromyces marxianus and form fertile recombinants at frequencies observed in intraspecific crosses, are accepted as varieties of Kluyveromyces marxianus. Hybridization was observed between Kluyveromyces marxianus var. lactis and the presumed imperfect forms of some Kluyveromyces species, namely Candida kefyr, Candida macedoniensis and Torulopsis sphaerica. Recombination was not detected in crosses involving Kluyveromyces marxianus var. marxianus and representatives of other yeast genera, i.e. Pichia, Saccharomyces, Torulaspora and Zygosaccharomyces. Conclusions regarding the relationship between members of the genus Kluyveromyces, reached on the basis of this investigation are compared with those reported by other workers, who based their investigations on phenotypic characteristics as well as on the determinations of mol % G+C and DNA-DNA homology studies.

The SUC2 locus of Saccharomyces cerevisiae encodes two forms of invertase; a constitutively expressed cytoplasmic enzyme and a glucose-repressible secreted and glycosylated enzyme which is initially produced with an amino-terminal signal peptide. The coding sequence of the SUC2 locus has been placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere based yeast plasmid vector from which invertase is expressed in a Sue" strain of yeast. Oligonucleotide-directed mutagenesis has been used to create a PstI site in the gene at the point encoding the signal peptide cleavage site. An internal methionine codon, the translation start for the cytoplasmic invertase, has been replaced by a serine codon. Mutants in the signal peptide sequence have been produced by replacing the region of the gene upstream of the PstI site with synthetic oligonucleotide cassettes with mixtures of nucleotides at several positions. The mutants could be divided into three classes based on their ability to secrete invertase. The first class of mutants produced secreted invertase, but in reduced amount. There is no obvious correlation between mutation and phenotype. The second class, represented by mutant 4-55B, also exhibited a reduced level of invertase, but a significant fraction (30%) of the enzyme is intracellular. This mutant had a delay in signal peptide cleavage which retards passage of invertase through the secretory pathway. The third class was defective in secretion. Most were defective in translocation from the cytoplasm to the lumen of the endoplasmic reticulum (ER), and produced enzymatically active, non-glycosylated pre-invertase in the cytoplasm. This class of mutant invertases, when transcribed and translated in vitro, was not processed by canine pancreas signal recognition particle (SRP) and microsomes. Comparison of the sequences of the mutant signal peptides of this non-translocating class identifies amino acids at the extreme amino-terminus as the causative defect. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The MAT∝ locus of the yeast Saccharomyces cerevisiae encodes two regulatory proteins responsible for determining the ∝cell type. The MAT∝1 gene encodes ∝1, a positive regulator of ∝cell-specific genes, whereas the MAT∝2 gene encodes a negative regulator of a cell-specific genes (∝2). MAT∝2. (in conjunction with the MATα1 gene) also determines the α/∝ diploid cell type by repressing haploid-specific genes. ∝2 exerts its effect at the transcriptional level in the ∝ cell by binding to a sequence located upstream of α cell-specific genes. The present study undertook to examine, through in vitro genetic manipulation, the structure/function relationship of the MAT∝ regulatory proteins, particularly∝2, in their role as gene regulators. The construction of mutant MAT∝2 genes containing termination codons at various points within the gene, and subsequent transformation of the mutant genes into mat∝2 yeast, indicated that the carboxy-terminal one-third of the gene product was necessary for full repressor activity in the haploid as well as in the diploid. A segment within the carboxy-terminal one-third of ∝2 displays some homology to the higher eukaryote homeo domain as well as to a prokaryotic bihelical DNA-binding structural motif. This region of the gene was subjected to semi-random missense mutagenesis in vitro and the mutant genes were analyzed by transformation into strains containing chimaeric genes that encode β-galactosidase from ∝2 and a1/∝2. repressible promoters. In this manner it was demonstrated that most of those residues in ∝2. which correspond to conserved amino acids in the prokaryotic DNA-binding structure and in the homeo domain are essential for the two repressor activities of ∝2. Several mutations more severely affected the ability of ∝2 to repress α-specific genes than haploid-specific genes. Analysis of the temperature dependence of the activities of some of the mutants was consistent with the existence of a helix-turn-helix structure at this region of the protein. Finally, further analysis of some of these mutants in vitro confirmed that the observed defect correlated with a loss of DNA-binding activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Thesis (MScAgric)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defence reactions comprising both constitutive and inducible barriers. While disease is the exception, such exceptions can be costly and even devastating. In particular, fungal diseases remain one of the major factors limiting crop productivity worldwide, with huge losses that need to be weighed up against massive cash inputs for pesticide treatments. Part of the defence reactions of plants is the synthesis of pathogenesis-related proteins, such as the plant hydrolases, glucanases and chitinases. In recent years, attention has been paid to the implementation of these proteins in plant transformation schemes. The rationale for this approach was that these antimicrobial agents not only degrade the main cell wall components of fungi, but also produce glucosidic fragments that act as elicitors of the biosynthesis of defence metabolites by the host. Furthermore, since these active antimicrobial agents are individually encoded by single genes, these defence systems should and have been shown to be highly amenable to manipulation by gene transfer. In this study, yeast glucanases from Saccharomyces cerevisiae were evaluated for their potential as antifungal proteins. The glucanases tested for their antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2 genes, encoding an exoglucanase and an endoglucanase respectively. An in vitro assay performed on these glucanases indicated that exoglucanase had a more detrimental effect on B. cinerea hyphal development and growth than the endoglucanase; the former caused typical disruption of the cells and leakage of cell material. The yeast exoglucanase was subsequently subcloned into a plant expression cassette containing the strong constitutive 358 promoter, yielding plasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted the exoglucanase to the apoplastic region with a signal peptide from an antimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 and pMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens to facilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration was confirmed with southern blot and PCR analyses for both the E and MJE lines. The expression and heterologous production of the EXG1-encoded exoglucanase in the E-transgenic lines was shown with northern blots and activity assays respectively. Moreover, the high level of expression of the yeast exoglucanase led to a decrease in susceptibility of the E lines to B. cinerea infection in comparison to the untransformed tobacco controls. An average decrease in disease susceptibility of 40% was observed in an in planta detached leaf assay. Crude protein extracts from the E lines were also analysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungal growth by average 20%, thus further confirming the antifungal nature of the yeast exoglucanase. Although integration of the MJ-EXG1 expression cassette was confirmed, no mRNA levels could be detected with northern blot or RT-PCR analysis of the MJE lines. These lines also did not show any in vitro antifungal activities or a decrease in susceptibility to B. cinerea infection in the detached leaf assay. It is suspected that this result is possibly linked to gene silencing, a phenomenon quite frequently associated with heterologous and/or overexpression of glucanases in plant hosts. It appears as if the targeted overexpression to the apoplastic space triggered the gene silencing response, since the intracellularly overexpressed product was produced and shown to display activity. The yeast exoglucanase thus joins the list of silenced glucanases in overexpression studies in plants. Overall, this study confirmed the antifungal characteristics of the Saccharomyces exoglucanase and provides valuable information of the possibility of utilising yeast glucanases in a transgenic environment. A decrease in the susceptibility of tobacco to B. cinerea infection, as shown by the overexpressed EXG1-encoded exoglucanases, merits further investigation into the use of this gene in the engineering of disease-resistant crops. / AFRIKAANSE OPSOMMING: Die weerstand van plante teen infeksie deur fitopatogeniese mikroórganismes is die resultaat van verskeie meervoudige verdedigingsreaksies wat beide konstitutiewe en induseerbare versperrings behels. Terwyl siekte die uitsondering eerder as die reël is, kan sulke uitsonderinge duur en selfs verwoestend wees. In die besonder is swamsiektes een van die vernaamste faktore wat gewasproduksie wêreldwyd beperk, met enorme verliese wat teen kontantinsette vir plaagdoders opgeweeg moet word. Deel van die verdedigingsreaksie van plante is die sintese van patogeen-verwante proteïene, soos die planthidrolases, -glukanases en -chitinases. In die onlangse tyd is aandag geskenk aan die implementering van hierdie proteïene in plant transformasieskemas. Die grondrede hiervoor was dat hierdie antimikrobiese agente nie net die hoof selwandkomponente van swamme kan afbreek nie, maar ook glukosidiese fragmente produseer wat as ontlokkers van metabolietbiosintese vir die verdediging van die gasheer kan optree. Aangesien hierdie aktiewe antimikrobiese agente individueel deur enkele gene enkodeer word, blyk hierdie verdedigingsisteme om hoogs ontvanklik vir manipulasie deur geenoordrag te wees. In hierdie studie is die gisglukanase van Saccharomyces cerevisiae vir hul potensiaal as antifungiese proteïene geëvalueer. Die glukanases wat vir hul antifungiese aktiwiteit teen Botrytis cinerea getoets is, was die gis EXG1- en -BGL2-gene, wat onderskeidelik vir "n eksoglukanase en 'n endoglukanase enkodeer. "n In vitro toets wat op hierdie glukanases uitgevoer is, het aangedui dat die eksoglukanase 'n meer skadelike effek op die hife-groei en -ontwikkeling van B. cinerea as die endoglukanase gehad het; eersgenoemde het die tipiese ontwrigting van die selle en die uitlek van selmateriaal tot gevolg gehad. Die gis-eksoglukanase is gevolglik in 'n plant uitdrukkingskasset wat die sterk konstitutiewe 35S promotor bevat, gesubkloneer, wat plamiede pEXG1 en pMJ-EXG1 opgelewer het. Die pMJ-EXG1-konstruk het die eksoglukanase na die apoplastiese gebied geteiken deur 'n seinpeptied vanaf "n antimikrobiese peptied van Mirabilisjalaba, Mj-AMP2. Die pEXG1- en pMJ-EXG1-konstrukte is in Agrobacterium tumefaciens gemobiliseer, wat die gevolglike tabaktransformasies gefasiliteer het wat die E en MJE transgeniese tabaklyne onderskeikelik gelewer het. Transgeen-integrasie is deur suidelike klad- en PKR-analises vir beide die E en MJE lyne bevestig. Die uitdrukking en heteroloë produksie van die EXG1-enkodeerde eksoglukanase is in die transgeniese E lyne deur noordelike klad en aktiwiteitstoetse onderskeidelik aangetoon. Verder het die hoë uitdrukkingsvlak van die gis-eksoglukanase tot 'n vermindering in die vatbaarheid van die E lyne vir B. cinerea-infeksie relatief tot die ongetransformeerde tabakkontroles gelei. 'n Gemiddelde vermindering in siektevatbaarheid van 40% is in 'n in planta verwyderde-blaartoets waargeneem. Ru proteïen-ekstrakte van die E lyne is ook in 'n in vitro kwantitatiewe swamgroeitoets geanaliseer en het in vitro swamgroei met tot gemiddeld 20% geïnhibeer, wat dus verder die antifungiese aard van die gis-eksoglukanase bevestig het. Alhoewel die integrasie van die pMJ-EXG1 uitdrukkingskasset bevestig is, kon geen mRNA-vlakke met die noordelike klad- of RT-peR-analises van die MJE-Iyne waargeneem word nie. Hierdie lyne het ook geen in vitro antifungiese aktiwiteite of 'n vermindering in die vatbaarheid vir B. cinerea-infeksie getoon nie, soos in die verwyderde-blaartoets uitgevoer is nie. Dit word vermoed dat hierdie resultaat moontlik aan geenstilmaking gekoppel is, 'n verskynsel wat gereeld met heteroloë- en/of ooruitdrukking van glukanases in plantgashere gekoppel word. Dit blyk dat die ooruitdrukking wat tot die apoplastiese ruimte geteiken is, tot die geenstilmaking-respons aanleiding gegee het, aangesien die intrasellulêre ooruitgedrukte produk gemaak is en aktiwiteit getoon het. Die gis-eksoglukanase word dus deel van die lys van stilgemaakte glukanases in die ooruitdrukkingstudies van plante. In die algemeen het hierdie studie dus die antifungiese kenmerke van die Saccharomyces eksoglukanase bevestig en waardevolle inligting oor die moontlike gebruik van gis-glukanases in 'n transgeniese omgewing verskaf. 'n Afname in die vatbaarheid van tabak vir infeksie deur B. cinerea, soos deur die ooruitdrukking van EXG1-enkodeerde eksoglukanase getoon is, verdien dus verdere ondersoek van die gebruik van hierdie geen in die skepping van siekteweerstandbiedende gewasse.