NCR-sensitive gene expression and regulation of nitrogen interconversion by VID30 in Saccharomyces cerevisiae

Van der Merwe, George K., (George Karel)1968- (2002-03)

Dissertation (PhD)--University of Stellenbosch, 2002.

Thesis

ENGLISH ABSTRACT: Saccharomyces cerevisiae uses the nitrogenous compounds in its environment selectively. The basis of this phenomenon is the transcriptional regulation of genes whose products are required for nitrogen catabolism. A rich nitrogen source represses the expression of genes required for the degradation of poor nitrogen sources via the action of the target of rapamyein (TOR) signaling cascade. If only a poor nitrogen source is available, these genes are derepressed. This process is known as nitrogen catabolite repression (NCR) or nitrogen regulation. The DALI and DAL4 genes of S. cerevisiae are transcribed divergently from the 829 bp intergenic region. The five known UASNTR elements (GATAI-5) were mutated in the full context of the intergenic promoter. All five elements are required for the transcriptional activation of DAL4. The two elements most proximal to DAL4 (GATA4 and GATA5) contributed the most and the one most distal (GATAI) contributed the least to its expression. In contrast, three of the five elements (GATA2-4) are required for DALI activation. In addition, analyses revealed that no single element is shared equally between these two genes. Predictions as to the function of known nitrogen-regulating elements based on their sequence and location proved to be inaccurate in some cases. Mutation analyses of the three UISALL elements present in the intergenic promoter region revealed that UIS8, which does not share a high degree of homology with the consensus UISALL sequence, is required the most for transcriptional induction of both DALI and DAL4. Also, UIS7, which shares the most similarity with the UISALL consensus sequence, has the phenotype of a repressor-like element when mutated. These observations therefore portray the opposite phenotypes of what was expected. We identified a regulator, Vid30p, which is required for the transcriptional response of S. cerevisiae in low ammonia conditions. Genetic analyses of the vid30/j, mutant indicate that Vid30p functions by regulating the expression of genes required for the production and degradation of glutamate. The transcription of VID30 is NCR-sensitive, highly induced by low concentrations of ammonia, and rapamycin-sensitive. In addition, the vid30/j, mutant is hypersensitive to rapamycin, indicating that this protein is, directly or indirectly, controlled by the TOR signaling pathway.

AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae het die vermoeë om stikstofbronne vanuit die omgewing selektief te benut. Die basis van hierdie verskynsel is die transkripsionele regulering van gene wat vir proteïene kodeer wat stikstof katabolisme bemiddel. 'n Goeie stikstofbron onderdruk die transkripsie van gene wat met die degradering van swak stikstofbronne gemoeid is. Hierdie onderdrukking word deur die teiken-van-rapamisien (TVR)-seintransduksiepad bewerkstellig. Wanneer slegs 'n swak stikstofbron beskikbaar is, word hierdie gene geaktiveer. Hierdie verskynsel staan as stikstofkatabolietonderdrukking (SKR) of stikstofregulering bekend. Die DALI- en DAL4-gene van S. cerevisiae word divergent vanaf 'n 829 bp intergeniese area getranskribeer. Vyf UASNTR-elemente (GATAI-5) is in die volle konteks van die intergeniese promotor gemuteer. Al vyf elemente word vir DAL4 transkripsionele aktivering benodig. Die twee elemente mees proksimaal tot DAL4 (GATA4 en GATA5) lewer die grootste bydrae tot DAL4-geenuitdrukking, terwyl die mees distale element (GATAI) die kleinste bydrae lewer. In teenstelling hiermee lewer slegs drie van die vyf elemente (GATA2-4) 'n noemenswaardige bydrae tot DALI se uitdrukking. Nie een van die vyf elemente lewer 'n gelykwaardige bydrae tot die uitdrukking van DALI en DAL4 nie. Voorspellings betreffende die bydrae van die onderskeie UASNTR-elemente tot die uitdrukking van die DALI- en DAL4-gene, gebaseer op die sekwens en die posisie van die element in die promotor, was meestal onakkuraat. Die drie U/SALL-elemente in die intergeniese area is gemuteer en toon dat U/S8, wat nie 'n groot mate van homologie met die U/SALL konsensus sekwens deel nie, die mees kritiese element vir transkripsionele induksie van beide DALI en DAL4 is. UIS7, wat 'n hoër mate van homologie met die UISALL konsensus sekwens deel, toon die fenotipe van 'n onderdrukkingselement wanner dit gemuteer word. Hierdie waarnemings is dus die teenoorgestelde van wat verwag is. Ons het 'n reguleerder, Vid30p, geïdentifiseer wat benodig word VIr die transkripsionele response van stikstofgereguleerde gene in lae konsentrasie ammonium. Genetiese analises van die vid3011 mutant toon dat Vid30p funksioneer deur die transkripsie van gene gemoeid met die vorming en degradering van glutamaat te reguleer. Die transkripsie van V/D30 is SKO-sensitief, word sterk deur lae konsentrasies ammonium geïnduseer, en is rapamisien-sensitief. Die vid30t!. mutant is ook hipersensitief vir rapamisien, wat aandui dat Vid30p, direk of indirek, deur die TVR-seintransduksiepad gereguleer word.

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