| Summary: | Dissertation (PhD)--University of Stellenbosch, 2002. === 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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