Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Abruptio placentae is the premature separation of the normally implanted placenta from the uterine wall, resulting in haemorrhage before delivery of the fetus. This has serious maternal and neonatal implications, and is one of the leading causes of perinatal and maternal mortality and morbidity in South Africa. Placental vasculopathies, such as abruptio placentae, are believed to result from faults occurring in early placental development. Placental protein 13 (PP13) is a member of the pregnancy-related protein family, and is believed to function in a number of important physiological processes such as trophoblast invasion, placentation and implantation. The aim of this study was to investigate whether DNA sequence variants in the LGALS13 gene (encoding PP13), underlie and/or confer susceptibility to abruptio placentae. The gene was screened and genotyped in a cohort of patients whose pregnancies were complicated by abruptio placentae, as well as an ethnically matched control cohort. Statistical and in silico analyses were performed in order to identify potential susceptibility factors in this South African cohort and to predict whether the identified variants may impact on gene expression or the structure and function of PP13. In addition, the luciferase reporter gene assay was employed to investigate the functionality of the -98A/C variant identified in the 5’ untranslated region of the LGALS13 gene. Statistically significant differences were observed between patient and control groups at the following loci in the Coloured population: -98A/C, IVS2 -36G/A, IVS2 -22A/G and the hotspot variant in exon 3 (p<0.05). These variants could represent a susceptibility profile of this population or alternatively have implications in the pathogenesis of abruptio placentae. The deletion of a single thymine in exon 3 was shown to result in truncation of PP13 and subsequent disruption of a number of cysteine residues and putative phosphorylation sites, which could impact on dimerization and ultimately, the function of the protein. The reporter gene assay revealed a significant reduction (p=0.004) in luciferase activity by the -98 C allele. iii In silico analysis suggests that this reduction could be due the disruption of a NF1 or GR transcription factor binding site. This study provides evidence that variants in the LGALS13 gene may underlie and/or confer susceptibility to abruptio placentae by impacting on gene regulation or resulting in the expression of an aberrant form of the PP13 which could affect functionality and thereby result in the disruption of normal implantation and placentation.

Thesis (PhD (Genetics))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Currently, South African aquaculture is dominated by the cultivation of Haliotis midae, which is estimated as the most lucrative sector of the industry, with 934 t being export in 2008, totalling an income of ZAR 268 million (40 million USD) in 2008. This represents 81% of the total rand value of the aquaculture sector. Abalone was the highest aquaculture commodity exported during the last two years from South Africa, representing 24% of the total tonnage exported. Employment in the aquaculture sector increased by approximately 80% between 2005 and 2008, and was highest in the abalone sector where the number of people employed increased by 234%. Despite these high production rates, no hatchery procedures have been developed specifically for H. midae. Most procedures and protocols currently used in South African abalone hatcheries have been adopted from cultivation methods used for foreign species. Although certain aspects of reproduction are universally conserved between abalones, it is important to consider the physiology and the origin of the species studied. To date, no scientific research has been conducted on the reproduction of H. midae, except for a few studies in the early 1990s, which focused on the basic reproduction of this species. No further studies have been done on H. midae reproduction under intensive culture. Currently, hatch-out rates obtained by most abalone farms in South Africa averages 80%, with a 50% settlement rate, and a final hatchery output of only 30%. This study reports on various aspects of H. midae reproduction that can influence its commercial culture. A detailed histological characterisation of gametogenesis was developed. Findings indicated that cultured H. midae reaches 50% sexual maturity at a shell width of between 25 mm and 30 mm. During fertilisation trials, a sperm concentration of 50 000 sperm mL-1 and egg concentrations lower than 50 eggs mL-1 produced the highest hatch-out rates. Whilst fertilisation volume did not influence fertilisation success, fertilisation potential of the eggs did decrease with time. Eggs older than 100 minutes showed a lower fertilisation potential than eggs fertilised earlier. A larval stress test was developed to evaluate larval resistance against chemical stress. It was determined that 50% of resultant larvae would exhibit morphological abnormalities after fertilised eggs were incubated in 0.7% dimethyl sulfoxide (Me2SO) for a 24 hour period. If larvae exhibited fewer abnormalities at this concentration, it could be deduced that the larvae had a high resistance to the negative effect of the toxicant, and could thus be seen as good quality larvae. When evaluating hybridisation potential between H. midae and H. spadicea, it was found that it was possible to fertilise spawned H. midae eggs with biopsied H. spadicea sperm. By incorporating the results obtained from the present study into current hatchery systems on South African abalone farms, higher hatchery yield could be achieved, which in turn would lead to an increase in commercial revenue. / AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse akwakultuur sektor word tans oorheers deur die produksie van Haliotis midae, en word gereken as die mees winsgewendste bedryf in die industrie, met 934 t uitgevoer in 2008, na-raming „n inkomste van ZAR 268 miljoen (40 miljoen Amerikaanse doller). Dit verteenwoordig sowat 81% van die totale rand waarde in die akwakultuur bedryf. Perlemoen was ook die grootste uitvoer kommoditeit gedurende die laaste twee jaar, en het tot sowat 24% van die totale uitvoer vanuit akwakultuur bygedra. Werksgeleenthede in die akwakultuur sektor het tussen 2005 en 2008 met ongeveer 80% gegroei, waarvan die hoogste groeisyfer in die perlemoen bedryf was, waar die aantal werknemers met 234% toegeneem het. Ten spyte van hierdie hoë produksie omset, is daar tans geen protokolle wat spesifiek vir die produksie van H. midae ontwerp is nie. Meeste van die tegnieke wat huidiglik gebruik word op Suid-Afrikaanse plase, is gebaseer op, en aangepas vanaf metodes daargestel in die internasionale bedryf vir uitheemse spesies. Alhoewel sekere aspekte van reproduksie tussen perlemoen spesies verband hou, is dit belangrik om die fisiologie en oorsprong van die spesie van belang, in ag te neem. Wetenskaplike navorsing gedoen op die reproduksie van H. midae is beperk tot studies in die vroeë 1990s, wat die basiese beginsels van die spesie se reproduksie ondersoek het. Daar is geen verdere studies op die reproduksie van H. midae, veral onder intensiewe teel toestande, gedoen nie. Tans toon die meeste perlemoen plase in Suid-Afrika „n produksie persentasie van ongeveer 80% larwes vanaf bevrugting, met „n afname na 50% met vestiging en „n gevolglike uitset van slegs 30%. Hierdie studie doen verslag oor verskeie reproduksie aspekte van H. midae wat die teel doeltreffendheid van perlemoen op kommersiële plase kan beïnvloed. „n Gedetaileerde histologiese karakterisering van gametogenese is ontwikkel. Daar is gevind dat geteelde perlemoen 50% geslagsrypheid bereik met „n skulp wydte van tussen 25 mm en 30 mm. Tydens bevrugting eksperimente is bepaal dat 50 000 sperm mL-1 en „n eier konsentrasie van laer as 50 eiers mL-1, die optimale gameet konsentrasies is vir effektiewe bevrugting. Alhoewel die volume water waarin bevrugting plaasvind nie „n invloed getoon het op bevrugtingsukses nie, is daar wel gevind dat die eiers se potensiaal om bevrug te word, afneem met verloop van tyd. Eiers ouer as 100 minute het „n verlaagde bevrugtingspotentiaal getoon teenoor eiers wat vroeër bevrug is. „n Larwale stres toets is ontwikkel om larwale weerstand teen chemiese stres te bepaal. Daar is gevind dat 50% van geproduseerde larwes morfologiese abnormaliteite sal toon indien bevrugte eiers vir „n periode van 24 uur in 0.7% dimetiel sulfoksied (Me2SO) geïnkubeer word. Indien larwes minder abnormaliteite toon by hierdie konsentrasie, beteken dit dat hierdie larwes meer weerstand kan bied teen die negatiewe effek van die toksiese middel, en dus beskou kan word as goeie kwaliteit larwes met hoë lewensvatbaarheid. Met die evaluering van hibridisasie potensiaal tussen H. midae en H. spadicea, is gevind dat dit moontlik is om vrygestelde H. midae eiers te bevrug met H. spadicea sperm wat verkry is deur „n biopsie. Die implementering van hierdie studie se bevindinge in kommersiële H. midae produksiesisteme sal daadwerklik bydra tot die optimisering van bestuurspraktyke en „n verhoging in die totale produksie doeltreffendheid van sulke sisteme.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Iron is an essential micronutrient that is an absolute requirement for correct cellular function in all eukaryotic organisms. However, ferrous iron has the ability to catalyze the formation of potentially toxic reactive oxygen species and regulation of iron metabolism is therefore of critical importance. Currently, there is little known about the co-ordinated regulation of the plethora of genes coding for proteins involved in this biochemical pathway, with the exception of the well characterized post-transcriptional IRE/IRP system. Regulation of gene expression in eukaryotic organisms is a highly intricate process. Transcriptional regulation is the first step and is controlled by the presence of specific cis-regulatory regions (cis-motifs), residing within the promoter region of genes, and the functional interactions between the products of specific regulatory genes (transcription factors) and these cismotifs. A combinatorial bioinformatic and functional approach was designed and utilized in this study for the analysis of the promoter architecture of genes of the iron metabolic pathway. The upstream non-coding region (~2 kb) of 18 genes (ACO1, CP, CYBRD1, FTH1, FTL, HAMP, HEPH, HFE, HFE2, HMOX1, IREB2, LTF, SLC11A2, SLC40A1, STEAP3, TF, TFRC, TFR2), known to be involved in the iron metabolism pathway, was subjected to computational analyses to identify regions of conserved nucleotide identity utilizing specific software tools. A subset of nine (CYBRD1, FTH1, HAMP, HFE, HFE2, HMOX1, IREB2, LTF, TFRC) of the genes were found to contain a genomic region that demonstrated over 75% sequence identity between the genes of interest. This conserved region (CR) is approximately 140 bp in size and was identified in each of the promoters of the nine genes. The CR was subjected to further detailed examination with comparative algorithms from different software for motif detection. Four specific cis-motifs were discovered within the CR, which were found to be in the same genomic position and orientation in each of the CR-containing genes. In silico prediction of putative transcription factor binding sites revealed the presence of numerous binding motifs of interest that could credibly be associated with a biological function in this pathway, including a novel MTF-1 binding site in five of the genes of interest. Validation of the bioinformatic predictions was performed in order to fully assess the relevance of the results in an in vitro setting. Luciferase reporter constructs for the nine CRcontaining genes were designed containing: 1) the 2 kb promoter, 2) a 1.86 kb promoter with the CR removed and 3) the 140 bp CR element. The expression levels of these three reporter gene constructs were monitored with a dual-luciferase reporter assay under standard culture conditions and simulated iron overload conditions in two different mammalian cell lines. Results of the luciferase assays indicate that the CR promoter constructs displayed statistically significant variation in expression values when compared to the untreated control constructs. Further, the CR appears to mediate transcriptional regulatory effects via an iron-independent mechanism. It is therefore apparent that the bioinformatic predictions were shown to be functionally relevant in this study and warrant further investigation. Results of these experiments represent a unique and comprehensive overview of novel transcriptional control elements of the iron metabolic pathway. The findings of this study strengthen the hypothesis that genes with similar promoter architecture, and involved in a common pathway, may be co-regulated. In addition, the combinatorial strategy employed in this study has applications in alternate pathways, and could serve as a refined approach for the prediction and study of regulatory targets in non-coding genomic DNA. / AFRIKAANSE OPSOMMING: Yster is ‘n noodsaaklike mikrovoedingstof wat ‘n vereiste is vir korrekte sellulêre funksie in alle eukariotiese organismes. Yster (II) of Fe2+ het egter die vermoë om die vorming van potensiële toksies reaktiewe suurstof spesies te kataliseer en dus is die regulasie van die yster metaboliese padweg van kardinale belang. Tans is daar beperkte inligting oor koördineerde regulasie van die gene, en dus proteïene waarvoor dit kodeer, in hierdie padweg. ‘n Uitsondering is die goed gekarakteriseerde na-transkripsionele “IRE/IRP” sisteem. Regulasie van geenuitdrukking in eukariotiese organismes is ‘n ingewikkelde proses. Transkripsionele regulasie is die eerste stap en word beheer deur die teenwoordigheid van spesefieke cis-regulatoriese elemente (cis-motiewe), geleë in die promotor area van gene, en die funksionele interaksies wat plaasvind tussen die produkte van spesifieke regulatoriese faktore (of transkripsie faktore) en hierdie cis-motiewe. ‘n Gekombineerde bioinformatiese en funksionele benadering was ontwerp en daarna gebruik in dié studie vir die analise van die promotor argitektuur van gene wat ‘n rol speel in die yster metaboliese padweg. Die stroomop nie-koderende streek (~2 kb) van 18 gene (ACO1, CP, CYBRD1, FTH1, FTL, HAMP, HEPH, HFE, HFE2, HMOX1, IREB2, LTF, SCL11A2, SLC40A1, STEAP3, TF, TFRC, TFR2), bekend vir hul betrokkenheid in die yster metabolisme padweg, was bloodgestel aan bioinformatiese analises om die streke van konservering te identifiseer met die hulp van spesifieke sagteware. Slegs nege (CYBRD1, FTH1, HAMP, HFE, HFE2, HMOX1, IREB2, LTF, TFRC) van die geanaliseerde gene het ‘n genomiese area bevat wat meer as 75% konservering getoon het. Hierdie gekonserveerde area (GA) is 140 bp in lengte en is geïdentifiseer in elk van die promotors van die nege gene. Die GA was verder bloodgestel aan analises, met die hulp van spesifieke sgateware, wat gebruik maak van vergelykende algoritmes vir motief karakterisering. Vier cis-motiewe is identifiseer en kom voor in dieselfde volgorde en oriëntasie in elk van die gene. In silico voorspelling van moontlike transkripsie faktor bindingsplekke het getoon dat daar talle bindingsmotiewe van belang teenwoordig is en dié motiewe kan gekoppel word aan biologiese funksies in hierdie padweg, insluitend ‘n nuwe MTF-1 bindingsplek in vyf van die gene van belang. Die bioinformatiese analises is verder gevalideer om die relevansie van die resultate in ‘n in vitro sisteem ten volle te assesseer. Luciferase rapporteerder konstrukte is vir die nege gene ontwerp wat die volgende bevat: 1) die 2 kb promotor, 2) ‘n 1.86 kb promotor met die GA verwyder en 3) die 140 bp GA element. Die vlakke van uitdrukking van hierdie drie rapporteerder konstrukte was genormaliseer met ‘n dubbele-luciferase rapporteerder assay onder standaard kultuur kondisies en gesimuleerde ysteroorlading kondisies in twee verskillende soogdier sellyne. Resultate van die luciferase assays dui aan dat die GA promotor konstrukte statisties betekenisvolle variasie toon in vergelyking met die onbehandelde kontrole konstrukte. Verder, die GA blyk om transkipsionele regulatoriese effekte te medieer via ‘n yster-onafhanklike meganisme. Dit blyk duidelik dat die bioinformatiese voorspellings ook funksioneel getoon kon word en was dus relevant in dié studie en regverdig verdere ondersoek. Hierdie eksperimentele ontwerp verteenwoordig ‘n unieke en omvattende oorsig van nuwe transkripsionele beheer elemente wat voorkom in die yster metaboliese padweg. Die resultate van dié studie versterk die hipotese dat gene met soortgelyke promotor argitektuur en wat betrokke is in ‘n gemene padweg saam gereguleer kan word. Daarbenewens, die gekombineerde strategie wat in hierdie studie gebruik is het toepassings in alternatiewe metaboliese paaie, en kan dien as ‘n verfynde benadering vir die voorspelling en studie van die regulerende teikens in nie-koderende genomiese DNS. / National Research Foundation (Thuthuka) / Stellenbosch University

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The World Health Organisation (WHO) has defined preterm labour as the onset of labour before 37 completed weeks of gestation with an incidence ranging between 5-10%. Although patient care has improved, the rate of preterm birth has slowly been increasing and currently impacts significantly on maternal and fetal mortality and morbidity. The complex condition of preterm labour involves multiple etiologies and risk factors, which complicates the search for candidate markers and / or biomarkers. The aim of this prospective study was to investigate potential genetic associations with preterm labour. The study cohort consisted of consecutive first-time booking, low-risk primigravid pregnant women from a restricted geographical region. The study cohort comprised 421 [306 Coloured and 115 Black] pregnant women presenting at the Paarl Hospital Obstetric clinic. Subsequently, DNA was extracted from whole blood and investigated for a range of known polymorphisms in pro-inflammatory and anti-inflammatory cytokines, as well as the novel LGALS13 gene, for potential variants that may impact on pregnancy outcome. Screening techniques involve combinations of allele-specific PCR amplification, Multiphor SSCP/HD analysis, restriction enzyme analyses and DNA sequencing. A significant association was demonstrated between the IL-1RN*2-allele and adverse pregnancy outcome, mainly in the preterm labour and hypertension group. The presence TNFα-308 A-allele was associated with overall adverse pregnancy outcome and preterm labour. In addition to this, a novel IL-1RN allele was identified in the control group. Mutation screening and subsequent statistical methods revealed an association between a novel LGALS13 exonic variant, 221delT, and preterm labour in Coloured women. Two previouslydocumented intronic variants (IVS2-22A/G and IVS3+72T/A) demonstrated linkage disequilibrium, signifying evolutionary conservation of exon three. Additionally, two novel intronic variants, IVS2-36 G/A and IVS2-15 G/A, demonstrated no association with adverse pregnancy outcome. In this study we identified rare novel exonic variants; two non-synonymous variants in exon three (M44V, [N=2] and K87R, [N=1]) and a silent variant in exon four (P117P, [N=1]) - all identified in individuals from the control cohort. Within coding exon three, an interesting variant [“hotspot”] was identified, which represents six polymorphic bases within an 11bp stretch. No associations were demonstrated with these variants and pregnancy outcome. Furthermore, a previously documented 5' “‘promoter” variant, -98 A/C, was identified and demonstrated no association with adverse pregnancy outcome. However, subdivision of lateonset pre-eclamptic cases revealed a significant association with the A-allele and late-onset preeclampsia. Genotype-phenotype investigation demonstrated association between the IL-10 -1082 A/G, IL-4 C/T and 221delT loci and poor pregnancy progress which manifested as (i) delivery of infants weighing <2000g, (ii) before 37 weeks of gestation. The findings of this study will strengthen our understanding of the pathophysiology underlying pregnancy complications and facilitate the further development of effective treatment strategies to reduce maternal and fetal morbidity and mortality. / AFRIKAANSE OPSOMMING: Die Wêreld Gesondheid Organisasie (WHO) klassifiseer voortydse kraam as kontraksie voor 37 volledige weke, met ‘n insidensie tussen 5-10%. Alhoewel pasiënte-sorg verbeter het, neem die tempo van voortydse geboorte steeds toe, wat ‘n groot impak het op moederstrefte en fetale mortaliteit en morbiditeit. Die komplekse kondisie van voortydse kraam sluit veelvoudige oorsake en risiko faktore in, wat die navorsing van kandidaat en / of biologiese merkers kompliseer. Die doel van hierdie prospektiewe studie, was die potensiële navorsing van genetiese assosiasies met voortydse kraam. Die studie kohort bevat opeenvolgende eerste bespreking van lae risiko primigravida swanger vrouens vanaf ‘n beperkte geografiese omgewing. Die studie kohort beslaan 421 [306 Kleurling en 115 Swart] swanger vrouens teenwoordig by die Paarl Hospitaal Verloskunde kliniek. Vervolgens was DNS geëkstraeer van bloedmonsters en geondersoek vir ‘n verskeidenheid van bekende polimorfismes in pro-inflammatoriese en antiinflammatoriese sitokiene, insluitend die nuwe sifting van die LGALS13 geen potensiaal vir variante wat ‘n impak op swangerskap uitkomste sal hê. Die siftings tegnieke toegepas, sluit in ‘n kombinasie van alleel-spesifieke amplifikasie, Multiphor enkelstring konformasie polimorfisme / heterodupleks analise, restriksie ensiem verterings en volgorde bepalings tegnieke. ‘n Betekenisvolle assosiasie was gedemonstreer tussen die IL-1RN*2-alleel en nadelige swangerskap, beperk tot voortydse kraam en die hipertensie groep. Die teenwoordigheid van die TNFα-308 A-alleel was geassosieer met algehele nadelige uitkomste en voortydse kraam. Daarby, was ‘n nuwe IL-1RN alleel geïdentifiseer in die kontrole groep. Mutasie sifting en opeenvolgende statistiese metodes, het ‘n assosiasie getoon tussen ‘n nuwe LGALS13 koderende variant, 221delT, en voortydse kraam in Kleurling vrouens. Twee voorafbeskryfde introniese variante (IVS2-22 A/G en IVS3+72 T/A), het ‘n betekenisvolle bewys opgelewer dat daar koppelings-onewewig bestaan tussen hierdie variante, en toon evolusionêre konservasie van ekson drie. Addisioneel was twee nuwe introniese variante ontdek, IVS2-36 G/A en IVS2-15 G/A, wat geen assosiasie getoon nie. In hierdie studie het ons ‘n nuwe seldsame koderende variante geïdentifiseer in die kontrole groep, waarvan twee nie-sinonieme variante was in ekson drie (M44V, N=2 en K87R, N=1) en ‘n stil variasie in ekson vier (P117P, N=1). Geleë in die koderende area van ekson drie, was ’n interessante variant [“hotspot’] ontdek, waarvan ses basisse in ‘n 11 basis paar area polimorfies is. Geen assosiasie was getoon met hierdie variante en swangerskap uitkomste nie. Verder was ‘n voorafbeskryfde 5' ‘promotor’ variant, -98 A/C, geïdentifiseer wat geen assosiasie getoon met nadelige swangerskap uitkomste nie. Onderverdeling van laat-aanvangs preeklampsie, het getoon dat die A-alleel ‘n betekenisvolle assosiasie getoon het met die ontwikkeling van laat pre-eklampsie. Genotipe-fenotipe interaksies het ’n assosiasie getoon tussen die IL-10 -1082 A/G, IL-4 C/T en 221delT lokusse en nadelige swangerskap uitkomste, wat manifesteer as (i) kraam van suigelinge wat <2000g weeg, (ii) geboorte voor 37 weke. Die bevindings van hierdie studie sal ons basiese kennis verbeter oor die patologie beskrywend aan swangerskap komplikasies, asook die fasilitering en ontwikkeling van effektiewe behandelings strategieë, om moederstrefte en fetale mortaliteit en morbiditeit te verminder.

Thesis (MSc (Genetics. Institute of Plant Biotechnology))--Stellenbosch University, 2008. / Sugarcane (Saccharum spp.) is an important crop which is cultivated worldwide for the high sucrose content in its stem. Conventional plant breeding has proven to be very successful over the years with regard to the enhancement of yield characteristics but due to the exhaustion of genetic potential in the commercial sugarcane germplasm recent progress has been slow. Genetic engineering seems to be a more attractive approach to enhance sucrose content and pest resistance in the stems but requires appropriate transgenes and suitable promoter. A promoter is essential to drive the transcription of a gene and is therefore critical to the success of transgenic approaches in sugarcane crop improvement. A negligible number of strong stem-specific promoters is available for use in sugarcane and this is one of the major limitations to genetic engineering. The goal of this project was to isolate a stemspecific promoter from maize and sorghum to drive stem-specific transgene expression in sugarcane. The approach used was to source promoters from non-sugarcane grass species with less complex genomes to simplify isolation and possibly counteract silencing. A cDNA sequence (SS) (EST clone, Accession number AW746904) from sugarcane was shown by Northern and Southern analysis to be stem-specific and to have an appropriately low copy number. The SS gene sequence was not expressed in the leaves of maize, sorghum or the sugarcane cultivars and prominent expression was observed only in the stems of the sugarcane hybrids N19 and 88H0019. The SS gene sequence was used to isolate its upstream regions from a Lambda genomic library of maize (Zea mays) and a sorghum (Sorghum bicolor) Bacterial Artificial Chromosome library (BAC). Of the four sorghum and six maize clones obtained in this study, a 4500 bp maize genomic DNA fragment (λ5) was sub-cloned in three fragments into separate pBluescript vectors using the ‘forced’ cloning approach for sequence and database (BLASTN) analysis. This revealed the complete SS gene sequence (975 bp), the promoter and a 300 bp intron region. A stretch of DNA sequence from nucleotides 664-3194 from the maize clone 5 sequence was designated the maize5-pro. Following sequence alignment of the maize and sugarcane promoter regions, significant sequence identity (68%) was observed between nucleotide 1675 and 3194 in maize and nucleotide 1506 and 2947 in sugarcane. The distance between the putative TATA-box and the TSS for this promoter (30 bp) was found to fall within the expected range of 32± 7 bp. The promoter region was analysed for possible cis-acting regulatory elements and revealed several promoter elements that are common in other plant promoters. The comparisons made between the putative transcription factors in maizepro-5 and the sugarcane promoter show that both promoter sequences are very similar as they share ten of the same transcription factors. However, the transcriptional factors WBOX, SRE and SP8BFIBSP8BIB are unique to the maize5-pro and the TAAG motif to the sugarcane promoter. Primers were designed with appropriate restriction sites and the promoter and intron (2850 bp) region was amplified by PCR (Polymerase chain reaction). The amplified fragment was fused inframe to the GUS reporter gene encoding β-glucuronidase to produce a transformation test vector. This will be used in future work to assess the functionality of the promoter through the production of stable transformants in which GUS activity can be measured in a range of tissues.

Thesis (MSc (Genetics))--University of Stellenbosch, 200 5. / The Lr19 translocation is associated with deleterious agronomic effects and as a result modified forms of the translocation have been derived by different researchers in an attempt to remove the genes responsible.

Thesis (MSc (Genetics))—University of Stellenbosch, 2007. / The porphyrias are a group of genetic disorders arising from mutations in either one of the final seven genes encoding the haeme synthesis enzymes. These disease-causing mutations lead to an enzyme deficiency that disrupts normal haeme production, resulting in clinical features due to the subsequent accumulation of porphyrin precursors. Like most of the porphyrias, variegate porphyria (VP) is characterized by high inter- and intra- familial clinical variability, with no apparent genotype-phenotype correlation. The delta-aminolevulinate synthase-1 gene (ALAS1) is an apparent candidate gene to explain the variable clinical expression observed in VP, since it encodes the first and rate-determining enzyme of haeme synthesis. Several studies have defined important regulatory elements for the human-, rat- and chicken ALAS1 gene that regulate expression patterns of this gene. It was hypothesized that in VP individuals, variants within/near critical regulatory sites might alter the transcription rate of this gene, and consequently increase/decrease the amount of haeme precursors accumulating as a result of the defective haeme synthesis enzyme. The aim of this study was to identify genetic variants that could influence gene expression in the proximal promoter area of the ALAS1 gene, as well as the two ALAS1-drug responsive enhancer sequences (ADRES) located further upstream. DNA (2133 bp per patient) of 19 clinically defined VP patients was analysed by polymerase chain reaction (PCR) and semiautomated DNA sequencing. Subsequently, in silico analyses using appropriate software programs, and in vitro studies using the luciferase reporter system, were performed to investigate the functionality of the identified variants on ALAS1 gene transcription...

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Oesophageal cancer (OC) is characterised by the development of malignant tumours in the epithelial cells lining the oesophagus. It demonstrates marked ethnic variation, with squamous cell carcinoma (SCC) being more prevalent in the Black population and adenocarcinoma (ADC) occurring more often in Caucasians. The aetiology of this complex disease has been attributed to a variety of factors, including an excess of iron (resulting in increased tumourigenesis), oesophageal injury and inflammation. The present study attempted to determine the mutation spectrum of the regulatory and coding regions of the ceruloplasmin (CP) gene, involved in iron metabolism, in the Black South African OC population. The patient cohort was comprised of 96 (48 male and 48 female) unrelated individuals presenting with SCC of the oesophagus. The control group consisted of 88 unrelated, healthy population-matched control individuals. The techniques employed for mutation detection in this study included polymerase chain reaction (PCR) amplification, heteroduplex single-strand conformation polymorphism (HEX-SSCP) analysis, restriction fragment length polymorphism (RFLP) analysis followed by bidirectional semi-automated DNA sequencing analysis to verify the variants identified. Mutation detection of CP resulted in the identification of fourteen previously described (5’UTR-567C→G, 5’UTR-563T→C, 5’UTR-439C→T, 5’UTR-364delT, 5’UTR-354T→C, 5’UTR-350C→T, 5’UTR-282A→G, V223, Y425, R367C, D544E, IVS4-14C→T, IVS7+9T→C and IVS15-12T→C) and four novel (5’UTR-308G→A, T83, V246A and G633) variants. Statistical analysis revealed that two of the novel variants were significantly associated with OC in this study; the promoter variant 5’UTR-308G→A (P=0.012) and the exonic variant G633 (P=0.0003). It is possible that these variants may contribute to OC susceptibility in the Black South African population. OC symptoms generally present late in the development of the disease, and as a result treatment after diagnosis is highly ineffective. Early detection of symptoms and subsequent treatment is therefore the most effective manner of disease intervention. In high incidence areas, such as the Transkei region of South Africa, the implementation of a screening programme would be the ideal way to achieve this goal. The information that can be gathered from the identification of potential modifier genes for OC can lead to improvements in early detection, which in turn may lead to advancements in the treatment and counselling to individuals with OC. To our knowledge, this is the first study concerning CP and its effects on iron dysregulation in the Black South African population with oesophageal cancer. / AFRIKAANSE OPSOMMING: Oesofageale kanker word gekenmerk deur die ontwikkeling van kwaardaardige gewasse in die epiteelweefsel van die oesofageale voering. Hierdie siekte demonstreer opvallende etniese variasie, met plaveisel selkarsinoom meer algemeen in die Swart populasie en adenokarsinoom meer algemeen in die Kaukasiese populasie. Die ontwikkeling van hierdie komplekse siekte word aan ‘n aantal faktore toegeskryf, insluitend ‘n oormaat yster (wat lei tot ‘n vermeerdering van gewasse) en oesofageale besering en -ontsteking. Die doel van die hierdie studie was om die mutasie spektrum van die regulatoriese- en koderingsarea van die ceruloplasmin (CP) geen, betrokke in yster metabolisme, in die Swart Suid Afrikaanse oesofageale kanker populasie te bepaal. Die pasiënt groep het bestaan uit 96 (48 manlik en 48 vroulik) onverwante individue met plaveisel selkarsinoom van die oesofagus. Die kontrole groep het uit 88 nie-geaffekteerde onverwante, populasie spesifieke individue bestaan. Die tegnieke aangewend vir mutasie deteksie in hierdie studie sluit in polimerase kettingsreaksie amplifikasie, heterodupleks enkelstring konformasie polimorfisme analise en restriksie fragment lengte polimorfisme analise, gevolg deur tweerigting semi-geoutomatiseerde DNS volgorde-bepalingsanalise om die geïdentifiseerde variante te bevestig. Mutasie deteksie van CP het tot die identifikasie van veertien reeds beskryfde (5’UTR-567C→G, 5’UTR-563T→C, 5’UTR-439C→T, 5’UTR-364delT, 5’UTR-354T→C, 5’UTR-350C→T, 5’UTR-282A→G, V223, Y425, R367C, D544E, IVS4-14C→T, IVS7+9T→C en IVS15-12T→C) en vier nuwe (5’UTR-308G→A, T83, V246A en G633) variante gelei. Statistiese analise het getoon dat twee van die nuwe variante betekenisvol geassosieerd was met oesofageale kanker in hierdie studie; die promotor variant 5’UTR-308G→A (P=0.012) en die eksoniese variant G633 (P=0.0003). Dit is moontlik dat hierdie variante mag bydra tot oesofageale kanker vatbaarheid in die Swart Suid Afrikaanse populasie. Oesofageale kanker simptome vertoon gewoonlik op ‘n latere stadium in die ontwikkelingsproses van die siekte, en as ‘n gevolg is behandeling na diagnose hoogs oneffektief. Vroegtydige identifikasie van die simptome en daaropvolgende behandeling is die mees effektiewe manier vir ingryping. In hoë voorkoms streke, soos die Transkei gebied van Suid Afrika, sal die implementasie van ‘n siftingsprogram die ideale manier wees om hierdie doel te bereik. Die inligting wat dan versamel word, insluitend identifisering van modifiserende gene vir oesofageale kanker, kan lei tot ‘n verbetering in vroegtydige deteksie van die siekte. In effek kan dit dan lei tot beter behandeling en berading vir individue met oesofageale kanker. So ver ons kennis strek, is hierdie die eerste studie wat CP en sy effek op yster disregulasie in die Swart Suid-Afrikaanse populasie met oesofageale kanker behels.

Thesis (MSc)--Stellenbosch University, 2012. / Triticale is an amphidiploid that consists of wheat (A and B) and rye (R) genomes. This cereal is fast becoming important on a commercial basis and warrants further assessment for the better management and breeding of the hybrid. The assessment of the genetic diversity among the wheat and rye genomes within triticale can be obtained by using molecular markers developed in both donor genomes. Simple sequence repeats markers (SSRs) and amplified fragment length markers (AFLPs) have been previously used to assess the genetic diversity among triticale lines. SSRs are highly polymorphic markers that are abundant and which have been shown to be highly transferable between species in previous studies while AFLP markers are known to generate plenty of data as they cover so many loci. Thus, the aim of this study was to develop a marker system suitable to assess the genetic diversity and relationships of advanced breeding material (and cultivars) of the Stellenbosch University’s Plant Breeding Laboratory (SU-PBL). Therefore, both AFLP and SSR markers were initially analysed using eight triticale cultivars (with known pedigrees) to facilitate cultivar identification. Fourty-two AFLP primer combinations and 86 SSR markers were used to assess the genetic diversity among the Elite triticale cultivars. The AFLP primer combinations generated under average polymorphism information content (PIC) values. Furthermore, these markers generated neighbour-joining (NJ) and unweighted pair group method with arithmetic average (UPGMA) dendograms that displayed relationships that did not correspond with the available pedigree information. Therefore, this marker system was found not to be suitable. A set of 86 SSRs previously identified in both wheat and rye, was used to test the genetic diversity among the eight cultivars. The markers developed in wheat achieved 84% transferability while those developed in rye achieved 79.3% transferability. A subset of SSR markers was able to distinguish the cultivars, and correctly identify them by generating NJ and UPGMA dendograms that exhibited relationships that corroborated the available pedigree data. This panel of markers was therefore chosen as the most suitable for the assessment of the advanced breeding material. The panel of seven SSR markers was optimised for semi-automated analysis and was used to screen and detect the genetic diversity among 306 triticale entries in the F6, Senior and Elite phases of the SU-PBL triticale breeding programme. An average PIC value of 0.65 was detected and moderate genetic variation was observed. NJ and UPGMA dendograms generated showed no clear groupings. However, the panel of markers managed to accurately identify all cultivars within the breeding program. The marker panel developed in this study is being used to routinely distinguish among the advanced breeding material within the SU-PBL triticale breeding programme and as a tool in molecular-assisted backcross.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Apoptosis represents a finely orchestrated and highly conserved natural form of cell death. It exhibits unique morphological and biochemical characteristics which culminate in the controlled dismantling of a cell from within followed by its discreet removal by phagocytic cells. Apoptosis is vital for the preservation of cell and tissue homeostasis but also performs several defensive and protective functions. Owing to its importance, apoptosis is highly regulated and a large number of proteins have been shown to mediate and safeguard the process. Furthermore, deregulated or altered levels of apoptosis can have severe pathological consequences; indeed, apoptosis has been shown to play a central role in several diseases, including neurological and autoimmune diseases as well as a variety of cancers. Consequently, the search for apoptotic-based therapies has received much attention and of vital importance to this quest is the characterisation of the specific mediators of apoptosis and their regulation as well as the identification of novel genes or proteins that can have a regulatory effect on apoptosis. It is thus the aim of this study to assist in this characterisation and also to identify novel candidate genes potentially involved in apoptosis. In a previously performed pilot study, three novel candidate genes potentially involved in apoptosis were identified by performing promoter-trap mutagenesis experiments. These genes were lipoic acid synthetase (LIAS), cyclophilin A (CYPA) and ribosomal protein L9 (RPL9). Since the methodology for this pilot study involved the use of functionally haploid cells, it was aimed in this study to verify these results in a diploid mouse cell line. Candidate gene knockdown was achieved by means of RNA interference and apoptosis assays were performed. A potential role for LIAS and CYPA in apoptosis was successfully verified in this study; however this could not be achieved for RPL9 and the gene was thus excluded from further study. In addition, nucleotide sequences isolated during the promoter-trap mutagenesis experiments in the pilot study were also investigated in order to identify additional novel candidate genes involved in apoptosis. By performing nucleotide BLAST searches, two potential candidate genes were identified, namely AHNAK nucleoprotein (AHNAK) and serum amyloid A-like 1 (SAAL1). Further bioinformatic analyses were performed with the four candidate genes in order to ascertain possible associations with apoptosis or cancer. Lastly, to further characterise the four candidate genes, the relative gene expression was investigated by means of quantitative PCR in two cancer and control cell lines. The results revealed significant differential expression for the majority of genes in the cancer cell lines when compared to the control cell lines. In conclusion, this study identified and characterised four novel genes potentially involved in apoptosis. Results obtained during this study can aid in the complete characterisation and functional annotation of these genes. Potential ties to apoptosis and associations with cancer are discussed for all four candidate genes and the possibilities of therapeutic strategies for anticancer treatments involving these candidate genes are noted. / AFRIKAANSE OPSOMMING: Apoptose verteenwoordig ‘n fyn georganiseerde en hoogs gekonserveerde natuurlike vorm van seldood. Dit vertoon unieke morfologiese and biochemiese eienskappe wat uitloop in die beheerde afbreek van ‘n sel vanuit die binnekant waarna dit onopsigtelik deur fagositiese selle verywder word. Apoptose is uiters belangrik vir die bewaring van sel en weefsel homeostase, maar dit vervul ook menigde afwerende and beskermde funksies. Vanweë sy noodsaaklikheid is apoptose hoogs gereguleer and ‘n groot aantal proteïene is al aangewys as bemiddelaars en beskermers van die proses. Verder, wangereguleerde en veranderde vlakke van apoptose kan ernstige patalogiese nagevolge hê; inderdaad, ‘n sentrale rol vir apoptose in verskeie siektes is al bevestig, insluitend neurologiese en outo-immuun siektes asook ‘n verskeidenheid van kankers. As gevolg hiervan ontvang die soektogte vir apoptose-gebaseerde terapieë vele aandag en uiters noodsaaklik vir hierdie soektogte is die karakterisering van die spesifieke bemiddelaars van apoptose en hul regulering asook die identifisering van nuwe gene of proteïene wat ‘n regulerende effek kan hê op apoptose. Dit is dus die doel van hierdie studie om by te dra tot hierdie karakterisering en ook om nuwe kandidaat gene wat moontlik betrokke kan wees in apoptose te identifiseer. In ‘n vorige loodsprojek is drie gene moontlik betrokke in apoptose geïdentifiseer deur middel van promoter-strik mutagenese eksperimente. Hierdie gene is lipoic acid synthetase (LIAS), cyclophilin A (CYPA) en ribosomal protein L9 (RPL9). Aangesien die metodiek in the loodsprojek gebruik gemaak het van funksionele haploïede selle, was dit die doel van hierdie studie om die resultate te bevestig in ‘n diploïede muis sellyn. Ribonukleïensuur (RNS) steuring is uitgevoer vir die uitklopping van die kandidaat gene en apoptose toetse is ook gedoen. Die bevestiging van ‘n moontlike rol vir LIAS en CYPA in apoptose was suksesvul in hierdie studie; alhoewel dit was nie bereikbaar vir RPL9 nie en hierdie geen is dus uitgesluit in verdere studies. Bykomend is nukleotied volgordes wat geïsoleer is tydens die promoter-strik mutagenese eksperimente in die loodsprojek ook nagesien om moontlike addisionele nuwe kandidaat gene te identifiseer wat moontlik betrokke kan wees by apoptose. Twee potensiële kandidaat gene, naamlik AHNAK nucleoprotein (AHNAK) en serum amyloid A-like 1 (SAAL1), was geïdentifiseer deur middel van nukleotied BLAST soektogte. Addisionele bioinformatiese analises is uitgevoer op die vier kandidaat gene om moontlike redes vir ‘n assosiasie met apoptose of kanker vas te stel. Laastens, om die kandidaat gene verder te karakteriseer, is daar ondersoek ingestel op die relatiewe geen uitdrukking van die kandidaat gene in twee kanker en twee normale sellyne. Die resultate het betekenisvolle differensiële regulering getoon vir meeste van die gene in die kanker sellyne in vergelyking met die normale sellyne. Ten slotte, vier kandidaat gene moontlik betrokke in apoptose is in die huidige studie geïdentifiseer en gekarakteriseer. Die resultate verwerf in hierdie studie kan moontlik bydra tot die volkome karakterisering en funksionele annotering van die kandidaat gene. Moontlike skakels met apoptose en assosiasies met kanker is bepreek vir die vier kandidaat gene en die moontlikheid van terapeutiese strategieë gebaseer rondom die kandidaat gene word ook genoem. / National Research Foundation (NRF)