Part of work presented in this thesis has been published: Regulation of gene expressions by GC-rich DNA cis-elements / J.P. Hapgood, J. Riedemann and S.D. Scherer in Cell biology international, vol. 25, 2001. / Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The sea urchin G-string binding factor 1 (suGF1) has previously been shown to bind with
high affinity and selectivity to stretches of contiguous deoxyguanosine residues, a DNA
motif found in the upstream regions of many unrelated genes from several organisms. It
has been proposed that suGF1 plays a role in transcriptional regulation.
Homopurine.homopyrimidine stretches have been shown to form unusual DNA structures,
in vitro. To investigate the potential of the suGF1 binding site to form unusual structures
under certain conditions, synthetic oligodeoxyribonucleotides containing the suGF1
poly(dG).(dC) binding site were subjected to circular dichroism (CD) analyses. The CD
results indicate that the suGF1 binding site forms a mixture of unusual DNA structures, as
deduced by comparison with the spectra obtained for B-DNA, triplex and quadruplex
conformations. These results are consistent with the hypothesis that suGF1 specifically
recognises G-strings that exhibit unusual structures.
Exhaustive database searches showed that suGF1 has no significant homology with any
previously identified proteins or cDNAs from any species. Given the relevance of
mammalian models to medical science, and since no sea urchin cell lines are currently
available, the identification of a mammalian functional homologue would facilitate
determination of the in vivo function of such a potentially important, putative, novel DNAbinding
protein in mammalian cell lines. In this study sequence analysis tools were used
to identify hORFX, a putative human functional homologue of suGF1. Similarities in the
domain organisation of the two proteins, prompted an investigation into the DNA-binding
properties of hORFX, as well as a more detailed structure prediction analysis, with a view
to determining whether hORFX is a functional homologue of suGF1. hORFX was
successfully expressed in vitro, but lacked the ability to specifically bind G-strings. Theoretical predictions suggest that suGF1 has a DNA-binding domain belonging to a
different family to that predicted for hORFX, consistent with differences in their respective
DNA-binding specificities. suGF1 and hORFX were predicted to have helix-turn-helix and
helix-loop-helix DNA-binding domains, respectively. Taken together the results do not
support the hypothesis that hORFX is a suGF1 homologue.
To date, no direct evidence for the in vivo function of suGF1 has been obtained. With a
view to performing transactivation assays in the future, the expression of suGF1 in yeast
was investigated in this project. An suGF1 expression construct was engineered and
transformed into a protease-deficient yeast strain. Nuclear extracts were prepared and
subjected to SOS-PAGE and electrophoretic mobility shift assays (EMSAs). suGF1 was
shown to be successfully expressed in yeast cells and exhibited similar G-string-binding
properties to that of native and in vitro transcribed and translated (IVT) suGF1. The
suGF1 eDNA was also subjected to in si/ico expression, which together with the SDSPAGE
results of yeast nuclear extracts and IVT suGF1, indicated that the protein might be
expressed as multiple truncated products, due to the utilisation of multiple AUG translation
start sites. These in vitro results are crucial for the ultimate outcome and correct
interpretation of future transactivation experiments and lay the foundation for further
investigation into the possible role of suGF1 in transcriptional regulation. / AFRIKAANSE OPSOMMING: In die verlede is bewys dat die seepampoentjie G-string-bindende faktor (suGF1) hoë
affiniteit en spesifisiteit vir aaneenlopende volgordes van deoksiguanosien residue besit.
Hierdie DNA motief kom algemeen voor in die stroom-op gebiede van verskeie gene in
verskillende organismes. Daar is 'n veronderstelling dat suGF1 betrokke is by die
regulering van geenuitdrukking.
Vroeër is bewys dat homopurien.homopirimidien-ryke areas die vermoë besit om in vitro
ongewone DNA-strukture te vorm. Die potentiaal van die suGF1-bindingsetel om
ongewone DNA-strukture te vorm is gevolglik deur sirkulêre dikroïsme (SD) analise
ondersoek. Vergelyking van die spektra vir B-DNA-, tripleks- en kwadrupleks-strukture
met dié van die suGF1-bindingsetel, toon duidelik dat laasgenoemde 'n mengsel van
ongewone DNA konformasies, onder die spesifieke eksperimentele omstandigehede,
aanneem.
Deeglike inspeksie van die beskikbare geen- en proteïendatabasisse vir alle spesies het
aangetoon dat suGF1 geen merkbare kDNA- of proteïenhomoloë besit nie. As gevolg van
die belang van soogdiermodelsisteme in die mediese wetenskappe, asook die
onbeskikbaarheid van seepampoentjie-sellyne, is 'n soektog na 'n funktionele suGF1
homoloog in soogdiere geloods. Die ontdekking van só 'n homoloog sal dit moontlik maak
om die rol van hierdie potensiaal belangrike en unieke DNA-bindingsproteïen te
ondersoek. Tydens hierdie soektog is spesiale analise-programme gebruik en 'n
potensiële menshomoloog van suGF1, hORFX, is geïdentifiseer. Die mees prominente
ooreenkoms tussen die twee proteïene is die soortgelyke rangskikking van funksionele
motiewe. Gevolglik is die DNA-bindings eienskappe van die hORFX-proteïen ondersoek, insluitende 'n detaileerde struktuur-funksie-voorspelling ten einde vas te stel of dit wél 'n
homoloog van suGF1 is. hORFX is suksesvol uitgedruk in vitro, maar besit nie die vermoë
om dieselfde G-string waaraan suGF1 spesifiek bind te herken nie. Teoretiese analise het
voorspel dat suGF1 en hORFX aan verskillende DNA-bindings proteïen-families behoort,
aangesien suGF1 'n heliks-draai-heliks en hORFX 'n heliks-lus-heliks motief bevat.
Hierdie inligting, tesame met die eksperimentele resultate, dui aan dat hORFX nie 'n
homoloog van suGF1 is nie.
Tot op hede is daar niks bekend aangaande suGF1 se funksie in vivo nie. Met die oog op
transaktiveringseksperimente in die toekoms, is die ekspressie van suGF1 in gisselle
tydens hierdie navorsingsprojek ondersoek. 'n suGF1 ekspressievektor is berei en gebruik
om 'n protease-negatiewe gissellyn te transformeer. Kernekstrakte is ondersoek deur
SDS-PAGE en elektroforetiese mobiliteitsessais. Daar is gevind dat suGF1 suksesvol
uitgedruk is in die gisselle. Die rekombinante suGF1 besit G-volgorde bindingsaktiwiteite
soortgelyk aan dié van suGF1 in kernekstrakte van seepampoentjies, asook in vitro
getranskribeerde-en getransleerde suGF1. Die kDNA vir suGF1 is ook in silico uitgedruk.
Tesame met die SDS-PAGE-resultate het laasgenoemde aangetoon dat die suGF1-kDNA
veelvuldige AUG-kodons bevat vir die inisiasie van proteïentranslasie. Dit lei moontlik tot
die translasie van 'n reeks proteïenprodukte wat verkort is aan die N-terminale kant,
afgesien van die volledige suGF1-proteïen. Die in vitro resultate in geheel is essensieel vir
die toekomstige uitvoering en interpretasie van transaktiveringseksperimente. Hierdie
projek lê gevolglik die fondasie vir 'n verdere ondersoek na die rol van suGF1 in die
regulering van geenuitdrukking.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52277 |
| Date | 12 1900 |
| Creators | Riedemann, Johann |
| Contributors | Hapgood, J. P., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 159 p. : ill. |
| Rights | Stellenbosch University |
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