published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Tο γονίδιο COUP-TF κωδικοποιεί έναν ορφανό υποδοχέα ο οποίος δρα ως μεταγραφικός παράγοντας και ανήκει στην υπεροικογένεια των υποδοχέων των στεροειδών/ θυρεοειδών ορμονών. Στον αχινό το γονίδιο COUP-TF εκφράζεται στο στοματικό έξώδερμα στο αναπτυξιακό στάδιο του γαστριδίου και στη βλεφαριδωτή ζώνη στο στάδιο του πλουτέα. Aργότερα κατά την ανάπτυξη της νύμφης εκφράζεται αποκλειστικά στα νευρικά κύτταρα και στον ενήλικα αχινό στις ωοθήκες, σε μυικά κύτταρα και άλλους ιστούς (Chan et.al. 1992). Στόχος των συγκεκριμένων πειραμάτων είναι η μελέτη της ρύθμισης του γονιδίου COUPTF στα αναπτυξιακά στάδια του γαστριδίου και του πλουτέα. Συγκεκριμένα, πραγματοποιήθηκε μια λεπτομερής ανάλυση της ανοδικής περιοχής του γονιδίου COUP-TF με σκοπό να καθοριστούν τα ιστοειδικά και χρονοειδικά στοιχεία που επηρεάζουν τη ρύθμιση. Προς το σκοπό αυτό αναγνωρίστηκε το σημείο έναρξης της μεταγραφής του γονιδίου. Kατόπιν, απομονώθηκε ανοδική περιοχή μεγέθους 2kb περίπου, από το +14 έως το -1930. Tο τμήμα αυτό κλωνοποιήθηκε σε φορέα που φέρει το γονίδιο αναφοράς GFP, καθώς και το βασικό υποκινητή του γονιδίου Endo16. Από το αρχικό αυτό κατασκεύασμα, δημιουργήθηκαν διαδοχικά ανοδικά ελλείμματα της περιοχής αυτής με τη μέθοδο της PCR. Eπιπλέον απομονώθηκαν επιμέρους μικρά ανοδικά τμήματα που δεν περιείχαν το βασικό υποκινητή του COUP-TF και κλωνοποιήθηκαν και αυτά στον ίδιο φορέα. Όλα τα κατασκευάσματα ενέθηκαν σε γονιμοποιημένα αυγά αχινού και μελετήθηκε το πρότυπο έκφρασης της πρωτεΐνης GFP στα στάδια του γαστριδίου και του πλουτέα. Tα αποτελέσματα έδειξαν ότι οι πρώτες 1079 βάσεις ανοδικά του +1 φέρουν τα απαραίτητα ρυθμιστικά στοιχεία για την έκφραση του COUP-TF στο στοματικό εξώδερμα και στο μεσέγχυμα. Eπιπλέον, η περιοχή από -232 έως -532 περιέχει έναν ενισχυτή της έκφρασης του COUP-TF στο στοματικό εξώδερμα ενώ το τμήμα από -1398 έως - 1639 έναν ενισχυτή της έκφρασης στο μεσέγχυμα. Ανοδικά του -1639 έως το -1930 περιέχεται ένας ισχυρός καταστολέας για την έκφραση του COUP-TF στο στοματικό εξώδερμα. Tα αποτελέσματα των ενέσεων με τα μικρά ανοδικά τμήματα επιβεβαιώνουν τα αποτελέσματα με τα διαδοχικά ανοδικά ελλείμματα και αναδεικνύουν ότι τα ρυθμιστικά στοιχεία που είναι απαραίτητα για την έκφραση του γονιδίου στο στοματικό εξώδερμα βρίσκονται στο τμήμα μεταξύ -232 και -1079. Η μελέτη ολόκληρης της ανοδικής περιοχής (έως -1930) δεν αναγνώρισε κάποιο τμήμα το οποίο να καταστέλλει την εκτοπική έκφραση του γονιδίου αναφοράς στα 7 μεσεγχυματικά κύτταρα. Η πραγματοποίηση κατασκευασμάτων που περιείχαν εκτός της ανοδικής περιοχής και την 5΄μη μεταφραζόμενη περιοχή οδήγησαν σε καταστολή της έκφρασης της GFP σε όλες τις κυτταρικές γραμμές. Η ανεύρεση στην 5’ μη μεταφραζόμενη περιοχή ενός στοιχείου απόκρισης για τον COUP-TF μας οδηγεί στην υπόθεση της πιθανής αυτορρύθμισης και συγκεκριμένα της αυτοκαταστολής του COUP-TF. / -

Eggs of many organisms contain a store of mRNA which supports protein synthesis during early embryonic development and various regions of the egg cytoplasm are not identical with respect to developmental potential. I investigated the extent to which sea urchin embryogenesis results from a progression of developmental events directed by the embryo, or an expression of a pre-formed maternal program. By the use of two-dimensional electrophoresis I demonstrated that cellular determination during embryonic development at the 16-cell stage is not accompanied by qualitative changes in the distribution within the embryo of abundantly-synthesized proteins, virtually all of which are coded by sequences present in the egg. Using two-dimensional gel electrophoresis, nucleic acid hybridization and molecular cloning, I demonstrated that there is restricted expression of paternal gene mRNA sequences in interspecies hybrid embryos. In some cases, this is due to a posttranscriptional perturbation of gene expression in the hybrid embryos.

No description available.

Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Thesis (doctoral)--Universität Bonn, 1893. / Cover title. At head of title: Mittheilung aus dem zoologisch. und vergleichend-anatomisch. Institut der Univ. Bonn." Vita. "Separat-abdruck aus: Zeitschrift für wissensch. Zoologie. Bd. LV. 4. Heft"--T.p. verso.

M.Comm. / Du Plessis (1993:649) states that the most common reasons for business failure are the lack of understanding of the market and inferior products. The first point in Timmons' criteria list focuses on the industry and market. This is of utmost importance as no correct analysis of a new venture's viability can be done if there is not a full understanding by the potential investor of the exact market that he/she intends to venture into. Timmons does not highlight the importance of the product as such in his criteria list although inferior products are one of the most common reasons for failure. Timmons' fatal flaw aspect, point six, is however very important as it is an "abort checkpoint". If the venture has a fatal flaw, regardless of everything else being perfect, it could cancel the potential venture. This fatal flaw aspect is in line with Cartland's "subjective determination" criteria. Various probabilities are difficult or impossible to quantify accurately. If the entrepreneur is not totally convinced of the probability of success, it should be seen as a fatal flaw and the venture abandoned. Timmons' list of eight criteria is seen to incorporate the broadest and most appropriate checklist. Chapter three will concentrate on the industry, market and product in more detail, thus ensuring a thorough understanding of the market and product, avoiding one of the weaknesses in Timmons' criteria. In chapter four the business opportunity for echinoderms in South Africa will be evaluated against the eight factors as outlined especially by Timmons in this chapter.

No description available.

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.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: South Africa's coastline is in the region of 3650kms and encompasses many different and dynamic marine environments. To enhance our current understanding of the population structure and gene flow patterns of intertidal zone marine species in this region, this study sets out to investigate the phylogeographic population structure of the Cape sea urchin, Parechinus angulosus, using mitochondrial and nuclear DNA sequence data collected in 2007 and 2008. Individuals were sampled from 18 geographic locations between southern Namibia and Durban, covering nearly the full extent of the species range. Sequence data were obtained from a 790bp region of the COI mtDNA gene (n=510) and a 182bp region of the nDNA SpREJ9 gene (n=145), respectively. The mtDNA data revealed 283 polymorphic sites (36%) defining 195 haplotypes, of which 160 were unique and 35 shared among individuals. Haplotype diversity (h) was found to be high both overall (h=0.95) and for individual localities (h=0.75-0.98), with nucleotide diversity (π) being low overall (π=0.013) as well as for individual localities (π=0.0033-0.0254). AMOVA revealed significant population structure among sampling sites in the Namaqua Province biogeographical region, as well as between three of the four respective coastal biogeographic provinces/regions. Gene flow was bi-directional among sampling sites in the south coast Agulhas and East Coast Province biogeographical regions, while gene flow in the Namaqua Province appears to be dominated by northwards movement. BAPS identified a significant break in the Cape Point region, which was also reflected in the gene flow patterns and parsimony networks. This broadly corresponds to previously identified biogeographic regions as well as genetic breaks for other marine species found along this coast. Fu's Fs statistics showed strong signal(s) of population expansion for individual sampling localities as well as for the data set as a whole, while MDIV estimated a time since expansion ranging from 7733-4759 years ago. The nDNA data revealed 54 variable sites (29.7%), defining 72 alleles of which 50 were unique and 22 shared among individuals. Many of the alleles (69.4%) were restricted to single sampling sites, with Betty's Bay on the south coast being the most diverse from a genetic viewpoint. Allelic diversity was high overall (h=0.86) while nucleotide diversity was low (π=0.025). No nuclear sub-group structure was identified by BAPS, although the parsimony network revealed shallow genetic structure between the Namaqua and Agulhas Provinces, with significant pairwise ФST values also recovered between their individual coastal localities. This points to at least one major barrier to gene flow for Parechinus angulosus along the South African coast, namely Cape Point. Several additional, smaller hindrances to gene flow along the coast were also identified, most of which are congruent with findings from studies on both other and sea urchin species. As a standalone study this research elucidated many aspects regarding the phylogeography of the Cape sea urchin, P. angulosus. However, it is when viewed in the broader context of invertebrate phylogeography along the southern African coastline that this research will provide its most critical insight. / AFRIKAANSE OPSOMMING: Geen opsomming