Regulation of the TPS1 gene expression in Saccharomyces Cerevisiae

Bihl, Georgia Gladys (1999-03)

Thesis (MSc)--University of Stellenbosch, 1999.

Thesis

ENGLISH SUMMARY: For many years the disaccharide trehalose was thought to be simply a carbon and energy reserve in the yeast Saccharomyces cerevisiae. However, a positive correlation between the trehalose content and thermotolerance, freeze tolerance, desiccation resistance, osmotolerance and exposure to toxic chemicals was observed in yeast as well as in other organisms. Therefore, trehalose is now considered to be a stress protectant rather than an energy reserve. This property of trehalose as a possible stress protectant has been used by the baking and brewing industries to increase the freeze tolerance of yeast. However, there is still a need in both industries to further optimise the freeze tolerance of yeast and to use the protective function of trehalose to the fullest extent. Genes encoding enzymes involved in the trehalose metabolic pathway have been manipulated in order to increase trehalose levels in yeast cells. However, it has been shown that higher trehalose levels do not necessarily increase the stress resistance of yeast. It therefore seems that factors other than high trehalose levels are responsible for stress resistance in yeast and these factors could function either in combination with or independently from trehalose. The focus of this study was to unravel the regulation of the TPS1 gene that encodes the first enzyme of the trehalose biosynthesis pathway, namely trehalose- 6-phosphate synthase. A deletion of the TPS1 gene leads to various phenotypes, including defects in trehalose synthesis and defective growth on glucose. This implies additional functions for the TPS1 gene and prompted us to investigate the transcriptional regulation of the gene. Based on previous results, two control mechanisms were investigated: an autoregulatory mechanism and regulation by stress responsive elements. Autoregulation implies that the gene product regulates expression of its own gene by interacting with the DNA sequences either within the promoter or within the open reading frame (ORF). The latter hypothesis was investigated by scanning the ORF for possible DNA-protein interactions and by expression studies with different truncation constructs of the TPS1 gene under glucose shift conditions. Our results, however, showed no evidence for an autoregulatory mechanism. The correlation between trehalose content and stress conditions led us to investigate the possibility of stress-regulated expression of the TPS1 gene. Stress responsive elements (STRE) have been identified in several stress-related genes where they confer stress-induced expression. Six putative STRE elements were identified in the promoter of the TPS1 gene. Gel retardation experiments and competition studies were used to show that nuclear proteins bind specifically to the STRE elements in the TPS1 gene promoter. Mutation analysis of these elements further showed that the STRE element closest to the ATG (STRE1) played a prominent role in regulating expression of the gene. However, this does not explain the various phenotypes observed for the TPS1 mutant. Further research is required to elucidate the various aspects concerning the control mechanisms involved in expression of the TPS1 gene.

AFRIKAANS OPSOMMING: Vir baie jare is die disakkaried trehalose as 'n energie- en koolstofbron in die gis Saccharomyces cerevisiae beskou. In Positiewe verband is egter tussen die trehalose-inhoud en weerstandbiedenheid teen hitte, vries, wateraktiwiteit (Aw) , osmotiese druk en blootstelling aan toksiese chemikaliee in giste sowel as in ander organismes, waargeneem. Oit het daartoe aanleiding gegee dat trehalose as 'n stresbeskermer eerder as In energiebron beskou is. Hierdie eienskap van trehalose as In moontlike stresbeskermer is deur die bakkers en brouers industriee benut om die vriestoleransie van giste te verhoog. Oaar is egter steeds in beide hierdie industriee 'n behoefte om die vriestoleransie van die gis verder te optimiseer om sodoende die beskermende funksie van trehalose ten volle te benut. Gene wat kodeer vir ensieme betrokke in die trehalose metaboliese weg, is reeds gemanipuleer om die trehalose konsentrasie in die gissel te verhoog. Oit is egter gevind dat hoer vlakke van trehalose nie noodwendig die stresweerstandbiedendheid van giste verhoog nie. Oit blyk dus dat ander faktore vir stres-weerstandbiedendheid in giste veranwoordelik is; hierdie faktore kan fn kombinasie of onafhanklik van trehalose funksioneer. Oie fokus van hierdie studie was om die regulering van die TPS1 geen wat die eerste ensiem in die trehalose-biosintetiese weg, (trehalose-B-fosfaat) enkodeer, te ontrafel. In Oelesie van die TPS1 geen lei tot verskeie fenotipes, insluitende defekte in trehalose sintese en defektiewe groei op glukose. Oit impliseer addisionele funksies vir die TPS1 geen en het daartoe aanleiding gegee dat ons die regulatoriese meganismes van die geen bestudeer het. Twee moontlike beheermeganismes is ondersoek: outoregulering en regulering deur middel van stres-beheerelemente bekend as STREs. Outoregulering impliseer dat die geenproduk uitdrukking van sy eie geen reguleer deur interaksie met ONA sekwensies in die promoter of in die oopleesraam (OLR). Laasgenoemde moontlikheid is ondersoek deur skandering van die OLR vir moontlike ONA-proteien interaksies en uitdrukking- studies met delesie-konstrukte van die OLR onder glukose kondisies" Geen bewyse vir outoregulering kon egter gevind word nie" Die verband tussen trehalose-inhoud en streskondisies het daartoe gelei dat ons die moontlikheid van stres-gereguleerde uitdrukking van die TPS1 geen ondersoek het. Stresbeheer-elemente (STRE) is reeds in ander stres-verwante gene ge"identifiseer waar dit stres-ge"induseerde uitdrukking bemiddel. Ses moontlike STRE-elemente is in die promotor van die TPS1 geen ge"identifiseer. Elektroforetiese mobiliteits-eksperimente en kompetisie-studies is aangewend om te toon dat nukluere prote"iene spesifiek aan die STRE-elemente in die TPS1 geenpromotor bind" Mutasie-analise van hierdie elemente het verder getoon dat die STRE-element naaste aan die ATG (STRE1). In prominente rol in die regulering van die geen speel. Hierdie resultate verduidelik egter nie die verskeie fenotipes wat vir die TPS1 mutant waargeneem is nie" Verdere navorsing word benodig om die verskillende aspekte van die regulatoriese meganismes betrokke in die uitdrukking van die TPS1 geen te verklaar.

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