Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The fission yeast Schizosaccharomyces pombe is able to effectively degrade extracellular
L-malate by means of a permease for the active transport of L-malate and a malic enzyme that
catalyses the intracellular oxidative decarboxylation of L-malate to pyruvate and CO2.
Sequence analysis of the S. pombe NAD-dependent malic enzyme gene, mae2, revealed an
open reading frame of 1695 nucleotides, encoding a polypeptide of 565 amino acids.
Mutational analyses of the mae2 promoter region revealed several putative cis-acting
elements. Two of these elements have homology with binding sites for eukaryotic cAMPdependent
regulatory proteins. The UAS I showed homology with the invert of the ADRI
binding site, an AP-2 binding site and the TGGCA element. The other putative cAMPdependent
site, UAS2, showed homology with the binding site for ATF/CREB and proved to
be a strong activator sequence that is required for expression of the mae2 gene. Three
negative acting elements, DRS I, DRS2 and DRS3 seem to function co-operatively to repress
transcription of the mae2 gene.
In this study northern and western blot analyses, as well as malic enzyme assays, showed
increased levels of mae2 transcription and enzyme activity when cells were grown under
fermentative conditions. The levels of mae2 expression increased approximately 4-fold in
30% glucose and 3-fold under anaerobic conditions. These increased levels of malic enzyme
may provide additional pyruvate for various metabolic processes when the mitochondria are
not fully functional under fermentative conditions.
The regulated expression of the mae2 gene was further investigated using mae2-1acZ fusion
plasmids that carried mutations in the DASI, UAS2 or the triple mutated DRSI/URS2/URS3
elements. These plasmids were transformed into S. pombe strains with mutations in the
cAMP-dependent or stress-activated signal transduction pathways to determine the signal for
the increased expression of the mae2 gene. The cAMP-dependent (Pkal ) and general stress
activated (Styl) pathways often act in parallel to regulate the activation of transcription
factors necessary for the expression of several S. pombe genes under different physiological
conditions. The results presented here suggest that regulatory proteins involved in the Pka l and Styl pathways play a role in the regulation of the mae2 gene under fermentative
conditions. Furthermore, some of the regulatory cis-acting elements in the mae2 promoter
may interact with these trans-acting factors to regulate the transcription of the gene under
different growth conditions. The mechanism of this interaction is not yet known and further
research is required to identify all the transcription factors involved in the regulation of the
mae2 gene. / AFRIKAANSE OPSOMMING: Die splitsingsgis S. pombe is in staat om ekstrasellulêre L-malaat effektief af te breek danksy
'n permease vir die aktiewe opname van L-malaat en 'n malaatensiem wat die intrasellulêre
oksidatiewe dekarboksilering van L-malaat na pirovaat en C02 kataliseer. DNA-geen
opeenvolgings van die NAD-afhanklike malaatensiemgeen, mae2, het 'n oopleesraam van
1695 nukleotiede getoon wat vir 'n polipeptied van 565 aminosure kodeer. Mutasie-analise
van die mae2-promoter gebied het verskeie moontlike cis-werkende elemente getoon. Twee
van die elemente toon homologie met bindingsetels vir eukariotiese cAMP-afhanklike
regulatoriese proteïene. Die DAS 1 toon homologie met die omgekeerde volgorde van die
ADRI bindingsetel, 'n AP-2 bindingsetel en 'n TGGCA element. Die ander moonlike cAMP
afhanklike setel, DAS2, toon homologie met die bindingsetel vir ATF/CREB en is 'n sterk
aktiveringselement wat vir die uitdrukking van die mae2-geen benodig word. Drie
onderdrukker-tipe elemente, DRSI, DRS2 en DRS3, funksioneer moontlik gesamentlik om
die transkripsie van die mae2-geen te onderdruk.
In hierdie studie het northern en western klad analise, sowel as malaatensiem aktiwiteitstoetse
verhoogde vlakke van mae2-transkripsie en ensiemaktiwiteit getoon wanneer die kulture
onder fermentatiewe toestande gegroei het. Die uitdrukking van die mae2-geen het ongeveer
4-voudig toegeneem in 30% glukose en 3-voudig onder anaërobiese toestande. Hierdie
verhoogde uitdrukking van die malaatensiem mag addisionele pirovaat vir verskeie
metaboliese behoeftes voorsien wanneer die mitochondria onder fermentatiewe toestande nie
volkome funksioneer nie.
Die uitdrukking van die mae2-geen is verder onder fermentatiewe toestande bestudeer deur
gebruik te maak van mae2-lacZ-fusie plasmiede wat mutasies in die moontlike DASI, DAS2,
of die drievoudig-gemuteerde DRS I/URS2/URS3 setels bevat. Hierdie plasmiede is in
S. pombe rasse met mutasies in die cAMP-afhanklike of stres-geaktiveerde seintransduksie
paaie getransformeer om die sein vir die verhoogde mae2-geen uitdrukking te bepaal. Die
cAMP-afhanklike (Pkal) en algemene stres-aktiverings (Styl) pad werk soms in parallel om
die aktivering van transkripsiefaktore betrokke in die uitdrukking van verskeie S. pombe gene onder verskillende fisiologiese toestande to bewerkstellig. Ons resultate dui daarop dat die
regulatoriese proteïene van die Pkal en die Styl paaie 'n rol in die regulering van die mae2-
geen onder fermentatiewe toestande speel. Daar is ook aanduidings dat sommige van die
regulatoriese cis-werkende elemente in die mae2-promoter wisselwerking met die transwerkende
faktore toon om die transkripsie van die geen onder verskillende groeitoestande te
reguleer. Die meganisme van hierdie interaksie is nog nie bekend nie en verdere navorsing is
nodig om al die transkripsiefaktore wat by die regulering van die mae2-geen betrokke is, te
identifiseer.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/51894 |
| Date | 03 1900 |
| Creators | Van der Merwe, Marizeth |
| Contributors | Viljoen, M., Bellstedt, D. U., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 148 p. : ill. |
| Rights | Stellenbosch University |
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