Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: In all cellular systems the ability to alter eellular programs in response to
extracellular cues is essential for survival. This involves the integration of
signals triggered by membrane bound receptors in order to adjust the
expression of target genes and enzyme activities and consequently
phenotypic outcome. The yeast Saccharomyces cerevisiae has evolved
several adaptations, such as, sporulation and pseudohyphal differentiation, in
order to survive changes in the surrounding environment. Pseudohyphal
differentiation and the related phenotype, invasive growth, are proposed to be
adaptations that enable the yeast to forage for scarce nutrients or escape
from a detrimental environment. This dimorphic transition is associated with a
change from the normal "yeast" form to a pseudohyphal form, which involves
changes in budding pattern, cell-cycle progression, cellular elongation, and
cell-eell and cell-substrate adherence. The outcome of these changes is
elongated eells, which bud in a unipolar fashion and do not separate after
budding to form chains of cells referred to as pseudohyphae. These
pseudohyphae are able to penetrate the surface of agar containing growth
medium, a process referred to as invasive growth.
Nutrient-induced adaptations, such as pseudohyphal growth, have been
extensively studied in S. cere visiae , and several factors have been implicated
in the regulation thereof, many of which are part of specific signalling
pathways. The most clearly defined are the filamentous growth specific MAP
kinase cascade and the Gpa2p-cAMP-PKA pathway. MUC1/FL011,
encoding a member of a family of cell wall associated proteins involved in cellcell/
cell-substrate adhesion, is regulated by these pathways and considered to
be critical in the establishment of pseudohyphal differentiation and invasive
growth. The promoter region of MUC1/FL011 represents one of the largest
yeast promoters identified to date, with cis-acting elements present up to 2.4
kb upstream from the first coding triplet. The upstream regulatory region of
MUC1/FL011 is almost identical to that of the STA2 gene, which encodes an
extracellular glucoamylase required for the utilisation of extracellular starch. As suggested by the extent of homology between these two promoters,
MUC1/FL011 and STA2 are co-regulated to a large degree and both require
the same transcription factors.
Mss11p plays a central role in the regulation of MUC1/FL011 and STA2 and
consequently starch metabolism and pseudohyphaI differentiation. The
regulation conferred by MSS11 on the transcriptional levels of MUC1/FL011
and STA2 also appears to be dependent on signals generated specifically in
the presence of low nitrogen and glucose. Mss11p does not have significant
homology to any other yeast protein, with the exception of limited homology to
the transcriptional activator F108p. However, several distinctive domains have
been identified in the MSS11 gene product. Firstly, Mss11p contains polyglutamine
and poly-asparagine domains. It also contains a putative ATP- or
GTP-binding domain (P-Ioop), commonly found in proteins such as kinases,
ATPases or GTPases. Two short stretches close to the N-terminal, labelled
H1 and H2, share significant homology to the transcriptional activator, F108p.
Both the H2 domain and the extreme C-terminal of Mss11p are able to
stimulate RNA polymerase II dependent transcription. Furthermore, the H1
domain together with the P-Ioop negatively regulates the activation potential
of the H2 domain.
This study presents further insight into the functioning of Mss11p and the
involvement of the separate activation and regulatory domains in mediating
transcriptional activation and pseudohyphal differentiation in response to
nutrient limitation. Genetic interactions between Mss11p and other factors
involved in the regulation of pseudohyphal growth and starch degradation
were revealed, and specific regions of Mss11p were shown to be required by
these factors in order to achieve their required function. In addition, results
obtained in this study implicates Mss11p in the regulation of Ca2+-dependent
flocculation and suggest that the FL01 gene is also regulated by Mss11p in
this capacity. / AFRIKAANSE OPSOMMING: Die vermoë om sellulêre programme in reaksie op ekstrasellulêre seine te
verander, is 'n essensiële vereiste vir alle sellulêre sisteme. Dit behels die
integrasie van seine gegenereer deur membraan-gebonde reseptore om
ekspressie van teikengene en ensiemaktiwiteite sodanig aan te pas dat
gewenste fenotipise uitkomste bewerkstellig kan word. Die gis
Saccharomyces cerevisiae het verskeie aanpassingsmeganismes ontwikkel,
soos byvoorbeeld sporulasie en pseudohifeforming, om veranderinge in die
omgewing te kan oorleef. Pseudohifevorming en die verwante fenotipe,
penetrasiegroei, word beskou as aanpassings te wees wat die gis in staat stel
om van 'n skadelike omgewing weg te kom, of dit in staat te stelom by skaars
voedingstowwe uit te kom. Hierdie dimorfiese transisie word geassosieer met
'n verandering van die normale "gisvorm" tot pseudohifevorming wat
veranderinge in die botpatroon, selsiklusprogressie, selverlenging, sel-sel en
sel-substraat aanhegting behels. Die uitkoms van hierdie verandering is
verlengde selle, wat unipolêr bot en nie van mekaar skei nie om sodoende
kettings van selle te vorm en waarna verwys word as pseudohifes. Hierdie
pseudohifes is ook in staat om die oppervlak van 'n agar bevattende
groeimedium te penetreer, 'n proses waarna verwys word as penetrasiegroei.
Aanpassings soos pseudohitevorminq is die afgelope dekade intensief
nagevors, en verskeie faktore en seintransduksienetwerke is in die regulering
daarvan geïmpliseer. Onder hierdie seintransduksienetwerke is die bes
gedefiniëerde paaie die filamentasie-spesifieke MAP-kinasekaskade en die
Gpa2p-cAMP-PKA pad. MUC1/FL011 kodeer vir 'n lid van 'n geenfamilie wat
met sel-sel/sel-substraat aanhegting geasosieer word en dit word deur hierdie
seintransduksie netwerke gereguleer. MUC1/FL011 word as essensieel vir
pseudohife vorming beskou. MUC1/FL011 word gereguleer deur die grootste
gispromoter wat tot op hede geïdentifiseer is, met cis-werkende elemente so
ver as 2.4 kb stroom-op van ATG. Die MUC1/FL011 promoter is feitlik
identies tot die van die STA2-geen, wat kodeer vir 'n ekstrasellulêre
glukoamilase wat die gis in staat stelom ekstrasellulêre stysel te benut. Weens die homologie tussen die twee promoters, word MUC1/FL011 en
STA2 tot In groot mate ge-koreguleer en beide benodig dieselfde
transkripsiefaktore.
Mss11p speel In sentrale rol in die regulering van MUC1/FL011 en STA2 en
dus ook in die regulering van pseudohifevorming en styselmetabolisme. Die
regulering wat deur Mss11p of MUC1/FL011 en STA2 uitgeofen word, blyk
verder onderhewig te wees aan seine wat gegenereer word spesifiek in die
teenwoordigheid van lae konsentrasies glukose en stikstof. Mss11p het nie
betekenisvolle homologie met enige ander gisproteïen nie, behalwe vir
beperkte homologie met die tranksripsionele aktiveerder F108p. Verskeie
onderskeidbare domeine is egter in die MSS11 geenproduk teenwoordig.
Eerstens, Mss11p bevat kenmerkende poliglutamien en poli-asparagien
domeine. Verder bevat Mss11p ook In voorspelde ATP- of GTP-bindings
domein (P-Ius), wat algemeen in proteïene soos kinases, ATPasaes en
GTPases voorkom. Twee kort areas naby die N-terminaal, aangedui as H1
en H2, het betekenisvolle homologie met die transkripsiefaktor F108p. Beide
die H2 domein en die ektreme C-terminaal van Mss11p is in staat om RNA
polimerase " afhanklike transkripsie te stimuleer. Verder het die H1-domein
in samewerking met die P-Ius In negatiewe uitwerking op die
aktiveringspotensiaal van die H2-domein.
Hierdie studie bied verdere insig tot die werking van Mss11p en die
betrokkenheid van die verskeie aktiverings- en reguleringsdomeine by die
oemiddetlinq van transkripsionele aktivering en pseudohifevorming in reaksie
op beperking van voedingstowwe. Genetiese interaksies tussen Mss11p en
ander faktore betrokke met die regulering van pseudohifevorming en
styselafbraak is in hierdie studie aangetoon. Voorts is daar ook gewys dat
spesifieke areas van Mss11p benodig word deur hierdie faktore om hulle
biologiese funksie uit te oefen. Daar is ook In rol vir Mss11p in die regulering
van Ca2+-afhanklike flokkulasie aangetoon en daar is bewys dat die FL01
geen deur Mss11p benodig word om hierdie effek uit te oefen.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50032 |
| Date | 03 1900 |
| Creators | Franken, Jaco (Cornelius Jacobus) |
| Contributors | Bauer, F. F., Pretorius, I. S., Patterton, H. G., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 125 p. : ill. |
| Rights | Stellenbosch University |
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