Functional analysis and recombinant expression of a sea urchin G-string binding factor

Riedemann, Johann (2001-12)

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.

Thesis

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.

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