Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The Russian wheat aphid (Diuraphis noxia, Kurdj., Hemipetra, Aphididae, RWA) is an important pest of wheat, causing large-scale damage and yield losses. Various studies have been done at a transcriptomics level, including complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs), suppressive subtractive hybridization (SSH) and micro-array, which have identified genes putatively involved in RWA resistance. Even though these candidate genes have been identified, their role in host defence still needs to be verified using a functional genetics approach. In this study virus induced gene silencing (VIGS) using a barley stripe mosaic virus (BSMV) vector, has been utilized to knock-down candidate genes of interest in a wheat cultivar with the Dn1-resistance gene (TugelaDN). In this study it was hypothesized that genes involved in the hypersensitive response (HR) may contribute towards resistance and were thus targeted for silencing. These include glutathione-S-transferase (GST), superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX). However, since aphid feeding also results in wounding, the genes were also analyzed under wounding only. Aphid fecundity is considered an indicator of involvement in RWA resistance, as susceptible plants result in higher aphid fertility. Findings in the study suggest that with wounding only, that Dn1 containing plants produce a greater hypersensitive response than susceptible controls. Ascorbate peroxidase was found to be important for wounding-induced resistance in Dn1 wheat plants. Under infestation conditions, silencing of superoxide dismutase Cu/Zn (SOD) and thylakoid-associated ascorbate peroxidase (tAPX) was found not to have an effect on aphid fertility and thus are not directly involved in resistance signaling. Knock-down of a phi-class glutathione-S-transferase F6 (TaGSTF6) transcripts however, had a large effect on aphid nymph numbers and thus may contribute to Dn1-resistance. Putative resistance genes silenced under aphid infestation conditions were a nucleotide binding protein (NBP) and resistance gene analogue 2 (RGA2). Analysis of NBP revealed its identity as a part of the iron homeostasis machinery in the cytosol, responsible for Fe-cluster assembly. Silencing of both NBP and RGA2 resulted in the expression of a susceptible phenotype. T10rga2-1A is an NBS-LRR protein known to be required for rust resistance in concert with resistance gene Lr10. T10rga2-1D silenced treatments resulted in susceptibility and plant death after aphid infestation, suggesting that T10rga2-1D may be a good up-stream candidate in Dn1-resistance. / AFRIKAANSE OPSOMMING: Die Russiese-koringluis (RWA) is ‘n pes wat ‘n belangrike ekonomiese invloed op koring opbrengste het en infestasie kan tot grootskaalse skade en oes verlies lei. Verskeie studies, onder andere komplimentêre DNA amplifiseerde fragment polimorfismes (cDNA-AFLPs), onderdrukkende onderskeidende hibridisaie (SSH) en mikro-reekse wat voorheen op transkriptomiese vlak gedoen is, het moontlike gene wat by RWA weerstand betrokke is, geïdentifiseer. Alhoewel hierdie gene reeds geidentifiseer was, hulle rol is nogtans onbekend. Dié gene moet nog getoets word, duur funksionele genetiese benaderingste maak. In hierdie studie is ‘n gars streep mosaïek virus vektor (BSMV) gebruik om kandidaat-gene van belang in ‘n Dn1-weerstandige geen-bevattende kultivar (TugelaDN) te onderdruk. Ondrukking van gene het deur middel van virus geïnduseerde geen onderdrukking (VIGS) plaasgevind. In hierdie studie is die hipotese gestel dat die gene betrokke by die hipersensitiewe reaksie (HR) ‘n invloed op plantweerstand kan hê en is dus geteiken vir geen-onderdrukking-studies. Hierdie gene het die volgende ingesluit: glutatioon-S-transferase (GST), superoksied dismutase Cu/Zn (SOD) en askorbien peroksidase (APX). Egter, omdat luisinfestasie ook tot verwonding aanleiding gee, is die onderdrukte gene ook onder alleenlik verwondingstoestande getoets. Luis vrugbaarheid is gebruik as indikator van betrokkenheid omdat meer vatbare plante ‘n hoër luis vrugbaarheid tot gevolg het. In die studie is gevind dat onder alleenlik verwondingkondisies, plante wat Dn1 bevat, ‘n groter hipersensitiewe respons vertoon, as vatbare kontroles. Daar is verder gevind dat askorbien peroksidase ‘n belangrike rol tydens verwondings-geïnduseerde weerstand in Dn1-plante speel. Daar is verder bevind dat die onderdrukking van superoksied dismutase Cu/Zn (SOD) en ‘n tilakoïed-geassosïeerde askorbien peroksidase (tAPX). Onder luis-infestasie kondisies, geen effek op luisvrugbaarheid gehad het nie en dus nie direk by die weerstandsrespons betrokke is nie. Die onderdrukking van ‘n phi-klas glutatioon-S-transferase F6 (TaGSTF6) het egter ‘n groot invloed op luis-vrugbaarheid gehad en kan dus ‘n rol in Dn1-weerstand speel. Die moontlike weerstands gene, geïdentifiseer as nukleotied bindings proteïen (NBP) en weestandsgeen anoloog 2 (T10rga2-1D), is getoets onder luis-infestasie kondisies. Die analise van NBP het getoon dat dit ‘n integrale deel van die yster homeostase meganisme in die sitosol, wat vir Fe-kluster samestelling verantwoordelik is, vorm. Onderdrukking van beide die NBP en T10rga2-1D het tot die uitdrukking van ‘n vatbare fenotipe aanleiding gegee. T10rga2-1A is ‘n NBS-LRR proteïen wat bekend is om noodsaaklik te wees tydens roes weerstandigheid in teenwoordigheid van die weerstandsgeen Lr10. T10rga2-1D-onderdrukte behandelings het tot vatbaarheid aangeiding gegee en daartoe gelei dat plante na luis-infestasies doodgaan. Hierdie resultate dui dus ‘n rol vir T10rga2-1D in Dn1-weerstandigheid aan, en suggereer verder dat hierdie geen ‘n goeie stroom-op kandidaat in Dn1-weerstandigheid is.

Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: To manipulate recombinant microorganisms for industrial processes, controllable genetic systems are needed that can coordinate expression of recombinant metabolic pathways. All components are sensitive to change and thus putative targets for modification and genetic elements and regulatory systems need to be understood and determined. Central in gene regulation is the transcription activators that mediate gene transcription mechanisms by binding to promoters in response to environmental signals. Promoter engineering entails the modification of transcription factors and their target promoters. In this study, a metabolic control system in Saccharomyces cerevisiae was constructed that would allow induction in response to physiological environment, specifically hypoxia and low temperature conditions. Two approaches were undertaken to find such a system. Firstly, a bi-directional reporter gene cloning vector was designed to search for novel hypoxiainducible promoters. Secondly, a transcription regulatory circuit was built, consisting of an inducible transcription regulator and promoter with a reporter gene through which it mediates transcription. Advantage was taken of the modular nature of proteins and functional domains originating from different transcriptional proteins were combined. A search for promoter elements sensitive to hypoxia from a S. cerevisiae genomic DNA (gDNA) library, using a bi-directional cloning vector, did not yield highly inducible promoters. It was concluded that a multitude of signals overlap, rendering genetic induction difficult to control. A synthetic regulatory system would minimize the impact of these multiple interactions. Such a genetic circuit was constructed, consisting of a chimeric transcription activator and a target fusion promoter. The chimeric transcription activator consisted of the GAL4 DNA binding domain, ADR1 TADIII transactivation domain and three domains of the MGA2 regulatory protein. The functional domains of Mga2p responsible for unregulated expression (at high basal levels) under both aerobic and hypoxia conditions were located, as well as a further upregulation under low temperature, and were mapped to the Nterminal and mid-Mga2p regions. A target fusion promoter consisting of a partial GAL10/1 promoter sequence and a Trichoderma reesei core xyn2 promoter were constructed as target for this chimeric transactivator. This synthetic promoter was fused to the T. reesei xyn2 open reading frame encoding for a readily assayable β-xylanase activity. Both the chimeric transactivator and fusion promoter-reporter gene cassettes were expressed from the same episomal plasmid, named pAR. Transformed into S. cerevisiae Y294, this regulatory system induced transcription under aerobic and hypoxia conditions. Furthermore, the reporter gene expression was upregulated by the chimeric transactivator at low temperatures. The chimeric transactivator mediated a seven-fold induction of the reporter gene under aerobic conditions in S. cerevisiae Y294 when transformed with plasmid AR. A two- to three-fold induction at 23ºC was reported under anaerobic conditions, relative to a reference strain expressing a transcription activator without the Mga2p domains. At 30ºC, a two- to three-fold induction under aerobic conditions and similar induction under oxygen-limited conditions were observed. Replacing the reporter gene with your favorite gene (for example a recombinant enzyme) and incorporating such a pAR system into a recombinant yeast should induce expression of the chosen gene under low temperatures, both aerobic and anaerobically (thus creating a controllable system). The system also has wider application in identifying other transcription factors’ signal-sensitive domains. The design of this system provides the ability to add a linker to a transactivator and to either create specific signal sensitivity or relieve the regulator of its signal dependence. It creates an easy system for assessing other transactivators and their domains with unknown functions and thus provides a ”workhorse and prospector in one”. / AFRIKAANSE OPSOMMING: Vir die manipulering van rekombinante mikroörganismes vir industriële prosesse word beheerbare genetiese stelsels benodig om gekoördineerde uitdrukking van rekombinante metaboliese weë teweeg te bring. Alle komponente van sulke stelsels is sensitief vir verandering en genetiese elemente en reguleerbare sisteme moet dus deeglik verstaan of bepaal word. Sentraal tot geenregulering is die transkripsie-aktiveerders wat geentranskripsie beheer deur aan promoters te bind in reaksie op eksterne omgewingsfaktore. Promotoringenieurswese behels wysigings van transkripsiefaktore en hul teikenpromotors. In hierdie studie is 'n genetiese beheerstelsel vir Saccaromyces cerevisiae ontwikkel wat induksie in reaksie tot spesifieke fisiologiese omgewingreaksies, naamlik hipoksie- en lae temperatuur, toelaat. Twee benaderings is gevolg: eerstens is ‘n tweerigting verklikker-geen vektor ontwikkel en gebruik om vir unieke induseerbare hipoksie-promoters te soek. Tweedens is ‘n transkripsie reguleringstelsel gebou wat uit ‘n induseerbare transkripsiereguleerder and promotor met ‘n verklikkergeen bestaan, waardeur transkripsie bemiddel kan word. Hierdie benadering benut die modulêre onderbou van proteïene en funksionele domeine afkomstig vanaf verskillende transkripsiefaktore is gekombineer. 'n Soektog na hipoksie-sensitiewe promotors vanuit 'n Saccharomyces cerevisiae-genoom- DNA (gDNA), deur van ‘n tweerigting verklikker-vektor gebruik te maak, het ongelukkig nie hoogs-induseerbare promotors opgelewer nie. Die gevolgtrekking was dat ‘n veelvoud van seine met mekaar oorvleuel en die beheer van genetiese induksie dus bemoeilik. Die ontwikkeling van ‘n sintetiese regulering-sisteem kan die impak van die veelvuldige interaksies verminder. Vir dié doel is ‘n sintetiese reguleringstelsel ontwerp, bestaande uit ‘n chimeriese transkripsie-aktiveerder met ‘n teiken fusie-promotor. Die chimeriese transaktiveerder bestaan uit die GAL4 DNA bindingsdomein, die ADR1 TAD III transaktiveringsdomein en drie domeine van die Mga2 reguleringsproteïen. In die studie is die funksionele domeins van Mga2p betrokke by lae temperatuur-respons en ongereguleerde uitdrukking (teen hoë basale vlakke) onder beide aërobiese en anaërobiese toestande aangedui en is tot die N-terminaal en middel-Mga2p areas gekarteer. ‘n Teiken-fusie-promoter, bestaande uit 'n gedeeltelike GAL1/10 DNA promotoropeenvolging en ‘n Trichoderma reesei kern xyn2-promoter, is as teiken vir hierdie chimeriese transaktiveerder saamgestel. Hierdie sintetiese promotor is aan die T. reesei xyn2 oopleesraam, wat vir ‘n maklik meetbare β-xylanase aktiwiteit kodeer, gekoppel. Beide die chimeriese transaktiveerder and fusie-promoter-verklikker-geenkaset word vanaf dieselfde episomale plasmied, bekend as pAR, uitgedruk. Hierdie reguleringsisteem induseer transkripsie onder aërobiese en hipoksie toestande in S. cerevisiae Y294. Verder word die verklikkergeen se uitdrukking deur die chimeriese transaktiveerder by lae temperature verhoog. Die chimeriese transaktiveerder induseer ‘n sewe-voudige induksie van die verklikkergeen onder aërobiese toestande by 23ºC vanaf die pAR-stelsel in S. cerevisiae Y294. ‘n Twee- tot drie-voudige induksie teen 23ºC is onder hipoksie toestande gevind, relatief tot induksievlakke van ‘n verwysingstam met ‘n transaktiveerder sonder die Mga2 domeine. By 30ºC is ‘n twee- tot drie-voudige induksie onder aërobiese en lae suurstofvlakke waargeneem. Deur die verklikker geen met ‘n jou-gunsteling-geen te vervang (bv. ‘n rekombinante ensiem) en so 'n pAR-sisteem in ‘n rekombinante gis te inkorporeer, word uitdrukking onder lae temperature onder beide aërobiese- en anaërobiese toestande geïnduseer (en sodoende word ‘n reguleerbare sisteem geskep). Die sisteem het wyer toepassing om sein-sensitiewe domeine van ander transkripsiefaktore te identifiseer. Die ontwerp van die stelsel maak dit moontlik om 'n skakel tot die transaktiveerder by te voeg wat óf sensitiwiteit tot 'n spesifieke sein skep, óf die reguleerder vanaf seinafhanklikheid verlos. So word ‘n bruikbare stelsel vir die bestudering van ander transaktivators en hul domeine met onbekende funksie geskep – ‘n “werksesel en prospekteerder in een”.

Poultry have many behavioural, structural and biological features that are ideal for domestication and for meat and egg production (Appleby et al., 1992). Because of the importance of poultry meat and eggs to the human population, breeders and farmers are always looking for ways of improving these traits. Artificial selection is the primary method of trait improvement, and involves selecting individuals with the highest breeding values as parents in each generation. There are a number of different methods of artificial selection, including: individual selection, between family selection, within family selection, family-index selection and index selection. In order to maintain a good response to selection breeders are constantly striving to improve the effectiveness and accuracy of the different methods of artificial selection for traits of economic importance. One method of achieving this goal is the use of computer models. Computer models can be used to simulate selection strategies and to predict what strategy will be the most appropriate for the improvement of a particular trait. This is important as all traits are influenced by many different genetic and environmental factors (Falconer and Mackay, 1996). This investigation was designed to compare the effectiveness of five different artificial selection strategies, namely individual selection, between family selection, within family selection, family index selection and index selection. Five computer models were developed using Microsoft Excel 2000 and these models were then used to compare the efficiencies of the five selection strategies for four different traits. The selection techniques were applied to an artificially, randomly generated population of 500 chickens. The four traits were egg weight with a heritability of 0.51, egg production with a heritability of 0.22, age at first egg with a heritability of 0.41 and body weight with a heritability of 0.55. Firstly, each of these traits were selected for independently using the first four selection methods and secondly the traits were selected for two at a time using index selection. The most significant results obtained from the single trait simulations were that for all traits family-index selection produced the best response to selection in the initial generations and between family selection produced the best response in the later generations. The traits with a higher heritability (egg weight and body weight) responded better to individual selection than they did to within family selection and between family selection in the initial generations. However, within family selection and between family selection proved to be more effective for traits with a low heritability such as egg production. Individual selection and family-index selection resulted in a very rapid decline in the standard deviation of all the traits. Between family selection resulted in the slowest drop in the standard deviation of all the traits, which is why this technique produced the best responses to selection in the later generations. The impact of the correlations between the economically important traits were evident from the results of index selection. For example, egg production is negatively correlated with egg weight making it difficult to gain a correlated response in both these traits simultaneously. Furthermore, egg production is negatively correlated with age at first egg implying that early maturing birds will lay more eggs, however, these eggs will be lighter. The majority of the results obtained were to be expected. Family-index selection takes all the information about an individual's breeding value into account resulting in this method of selection consistently identifying the most desirable individuals being selected. It is therefore the preferred method of selection under all circumstances. It is, however, often not economically and practically efficient to incorporate this technique and the use of another method of selection usually proves to be more beneficial. Individual selection proved to be most effective when applied to traits with high heritabilities, due to the fact that this method selects individuals based on their own phenotypic values. For traits with a high heritability, an individual with a good phenotypic value will have a good breeding value. Between family selection and within family selection proved better for traits with lower heritabilities. For traits with a low heritability the phenotypic value of an individual is a poor indicator of its breeding value. Information from a number of relatives may thus improve the accuracy of prediction of the breeding value by accounting for the influence of environmental effects. The use of computer models to simulate the selection techniques proved very successful in illustrating the effectiveness of the different selection techniques under various genetic and environmental conditions. The models may also prove to be very effective from an educational perspective. / Thesis (M.Sc.)-University of Natal, Pietemaritzburg, 2003.

Genetics has been identified as a subject area which many students find difficult to comprehend. The researcher, who is also a lecturer at the University of KwaZulu-Natal, had noted over a number of years that students find the field of quantitative genetics particularly challenging. The aim of this investigation was two-fold. Firstly, during the diagnostic phase of the investigation, to obtain empirical evidence on the nature of difficulties and alternative conceptions that may be experienced by some students in the context of quantitative genetics. Secondly, to develop, implement and assess an intervention during the remediation phase of the study which could address the identified difficulties and alternative conceptions. The research was conducted from a human constructivist perspective using an action research approach. A mixed-method, pragmatic paradigm was employed. The study was conducted at the University of KwaZulu-Natal over four years and involved third-year students studying introductory modules in quantitative genetics. Empirical evidence of students' conceptual frameworks, student difficulties and alternative conceptions was obtained during the diagnostic phase using five research instruments. These included: free-response probes, multiple-choice diagnostic tests, student-generated concept maps, a word association study and student interviews. Data were collected, at the start and completion of the modules, to ascertain the status of students' prior knowledge (prior knowledge concepts), and what they had learnt during the teaching of the module (quantitative genetics concepts). Student-generated concept maps and student interviews were used to determine whether students were able to integrate their knowledge and link key concepts of quantitative genetics. This initial analysis indicated that many students had difficulty integrating their knowledge of variance and heritability, and could not apply their knowledge of quantitative genetics to the solution of practical problems. Multiple-choice diagnostic tests and interviews with selected students were used to gather data on student difficulties and alternative conceptions. The results suggested that students held five primary difficulties or alternative conceptions with respect to prior knowledge concepts: (1) confusion between the terms variation and variance; (2) inappropriate association of heterozygosity with variation in a population; (3) inappropriate association of variation with change; (4) inappropriate association of equilibrium with inbred populations and with values of zero and one; and, (5) difficulty relating descriptive statistics to graphs of a normal distribution. Furthermore, three major difficulties were detected with respect to students understanding of quantitative genetics concepts: (1) students frequently confused individual and population measures such as breeding value and heritability; (2) students confused the terms heritability and inheritance; and, (3) students were not able to link descriptive statistics such as variance and heritability to histograms. Students found the concepts of variance and heritability to be particularly challenging. A synthesis of the results obtained from the diagnostic phase indicated that many of the difficulties and alternative conceptions noted were due to confusion between certain terms and topics and that students had difficulty with the construction and interpretation of histograms. These results were used to develop a model of the possible source of students' difficulties. It was hypothesized and found that the sequence in which concepts are introduced to students at many South African universities could be responsible for difficulties and alternative conceptions identified during the study, particularly the inappropriate association of terms or topics. An intervention was developed to address the identified difficulties and alternative conceptions. This intervention consisted of a series of computer-based tutorials and concept mapping exercises. The intervention was then implemented throughout a third year introductory module in quantitative genetics. The effectiveness of the intervention was assessed using the multiple-choice diagnostic tests and interview protocols developed during the diagnostic phase. The knowledge of the student group who participated in the intervention (test group) was compared against a student group from the previous year that had only been exposed to conventional teaching strategies (control group). t-tests, an analysis of covariance and a regression analysis all indicated that the intervention had been effective. Furthermore, an inductive analysis of the student responses indicted that most students understanding of the concepts of variance, heritability and histograms was greatly improved. The concept maps generated by students during the remediation phase, and data from the student interviews, provided an indication of the nature and extent of the conceptual change which had occurred during the teaching of the module. The results showed that most of the conceptual change could be classified as conceptual development or conceptual capture and not conceptual exchange. Furthermore, it seemed that conceptual change had occurred when considered from an epistemological, ontological and affective perspective, with most students indicating that they felt they had benefited from all aspects of the intervention. The findings of this research strongly suggest an urgent need to redesign quantitative genetics course curricula. Cognisance should be taken of both the sequence and the manner in which key concepts are taught in order to enhance students' understanding of this highly cognitively demanding area of genetics. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

Host genetic factors may partially account for the uneven distribution of HIV infection worldwide. In addition to influencing relative susceptibility to HIV, host genetic factors may also affect the rate of disease progression in persons who are already HIV infected. J.L-10 was previously identified as an AIDS restricting gene (ARG), i.e. human genes with polymorphic variants that influence the outcome of HIV-1 exposure or infection. IL-10 is a Th2 cytokine, with anti-inflammatory properties, and plays a significant role in the regulation of immune responses; this cytokine may also directly influence viral replication. This study focused on the role of genetic polymorphisms in the proximal promoter region of the IL-10 gene on HIV-:eptibility and primary HIV-1 pathogenesis in a South African comprising of women at high risk of HIV-1 infection In this study 228 black females from the CAPRISA Acute Infection cohort were genotyped for two polymorphisms that naturally occur within the proximal region of the IL-10 promoter, at positions -.1082 and -592 (tracking -819) relative to the transcription start site. DNA samples from study participants were genotyped using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method, which utilises specifically designed primers to detect single nucleotide polymorphisms. The allele frequencies for the mutant -1082G and -592A variants were 0.3203 and 0.333 respectively.Individuals homozygous for the mutation at the -392 position (AA genotype) were 2.78 times more likely to become HIV infected, compared to those who were homozygous wild type (CC genotype) at the same position (p-value=0.0237). Among those who became HIV infected, we found a hierarchical association between IL-10 promoter variants and HIV-1 plasma viral load or CD4+ T cell counts over the course year of HIV-1 infection. At earlier time points, i.e. 0-3 months post-te -1082GG group had significantly higher median viral loads than the -AA or -1082AG groups (pvalues= <0.0001 and 0.0003 respectively); and the -1082AA group had the highest median CD4'' T cell count compared to the -1082AG or -1082GG groups and this was significant (p-values= 0.0194 and 0.0122 respectively). At 6-12 months post-infection the median viral load of the -1082GG group was lower than -1082AA group, however this was not significant (p-value=0.6767). Analysis of the effect of the -592 polymorphism showed that the -592AA group had a lower median viral load at 0-3 months post-infection compared to the homozygous wild-type group (i.e. -592CC p~value=0.0093); and the median CD4+ T cell count for the -592AA group was significantly higher than the -592CC group (p~ value= 0.0198). At 6-12 months post-infection, the median viral load as well as the median CD4+ T cell count of the -592 A A group were both no longer significantly different to the -592CC group (p-values= 0.644land 0.6461 respectively). Plasma IL-10 expression was not significantly different between the IL-I0 genotypes for any of the polymorphic positions.Overall, these results suggest that polymorphisms within the IL-10 promoter may influence the risk of HIV infection and that they may affect primary HIV-1 pathogenesis. Interestingly, our data suggests that the effect of these polymorphic variants on viral and CD4+ T cell counts may vary according to time post-infection. To our knowledge, this is the first study to suggest that an ARG may have a differential effect on markers of disease progression depending on the phase of infection studied. The mechanisms underlying these observations require further studies and may have important implications for HIV/AIDS pathogenesis and the development of effective vaccine and immunotherapeutic strategies. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

The rate at which bacterial pathogens are becoming resistant to antibiotics is quite alarming, and therefore much attention has been focussed on this area. The mechanism whereby the bacterial cells acquire resistance is studied in order to determine how this process works as well as to determine if any future resistance mechanisms can be circumvented. In this study three different genera and the antibiotics that are resistant to them were used, namely, penicillin resistant Streptococcus, vancomycin resistant Enterococcus and methicillin resistant Staphylococcus. The results prove that the active sites SXXK, SXN and KT(S) G in the penicillin resistance Streptococcus plays a major role in resistance. It is seen in this study that the SXXK active site is found in all the resistant and most of the intermediate strains, therefore proving to be an important component of the cell wall resistance. It was subsequently noticed the greater the number of mutations found in the sequences the higher the resistance. Three dimensional structures showed the actives sites and their binding pockets. The results also show the change in conformation with a mutation in the active site. The results also proved that the Penicillin Binding Protein (PBP) genes essential for resistance are PBP Ia, PBP 2b and PBP 2x. The results obtained, for the vancomycin resistance in Enterococcus study, proved that the VanC and VanE cluster are very much alike and VanE could have evolved from VanC. There is also close similarity between the different ligase genes. The VanX 3D structure shows the position of the critical amino acids responsible for the breakdown of the D-Ala-D-Ala precursors, and the VanA ligase 3D structure shows the amino acids responsible the ligation of the D-Ala-D-Lac precursors. The analysis performed on the methicillin resistance in Staphylococcus study showed that the genes used to confer resistance are very similar between different strains as well as different species. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Please open electronic version for Abstract. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.

Maize (Zea mays L.) is an important agronomic crop with the maize industry forming an important component of the South African economy. Considerable effort has been directed towards the genetic improvement of maize through both conventional breeding and biotechnology. Genotype identification by DNA fingerprinting is becoming an important activity in plant breeding. A widely used molecular based and relatively inexpensive method for DNA fingerprinting is the randomly amplified polymorphic DNA (RAPD) technique. The RAPD technique was tested in this study for its potential use in maize breeding programmes. Initial results using the technique showed a low degree of reproducibility, therefore both the DNA isolation and RAPD protocols were extensively optimised. DNA quality and quantity, and choice of Taq polymerase buffer were three of the variables found to be influential in ensuring reproducibility. The ability of the RAPD technique to characterise seven maize genotypes was evaluated. Sixty random oligonucleotide primers were screened. Forty two primers scored a total of 233 fragments (an average of 5.5 per primer), but not all primers gave reproducible profiles. Eighteen primers scored a total of 110 loci for the presence (1) and absence (0) of DNA fragments. RAPD markers were able to distinguish between all seven genotypes with five primers producing specific fragments for four genotypes. Genetic similarity matrices were calculated using two software programmes i.e. Genstat 5™ release 4.1 (1993) and PAUP (Phylogenetic Analysis Using Parsimony) 4.0 beta version (Swafford, 1998). Cluster analysis was used to generate dendrograms to visualise the genetic relationships of the seven maize genotypes (only minor differences were observed between the Genstat or PAUP method of analysis). Genetic diversity ranged from 0.62 to 0.96. The estimation of genetic relationship was in accordance with the presumed pedigree of the genotypes showing that the RAPD technique demonstrates potential for genome analysis of maize. The applicability of the technique for marker assisted selection was also evaluated. Near-isogenic lines (NILs) for leaf blight (Helminthosporium spp.) were screened for polymorphisms using a total of 120 primers. Ten primers identified polymorphisms between the NILs. Four primers produced five polymorphic fragments present in the resistant inbred K0315Y and absent in the susceptible inbred D0940Y. A small F2 population of 14 individuals was produced by selfing the F1 of a cross between K0315Y and D0940Y. To speed up the generation time, the F1 and F2 plants were cultured by embryo rescue from 18d old harvested seed. One fragment of 627 base pairs produced by primer OPB-01 (5' GTTTCGCTCC 3') showed a 3: 1 segregation in the small F2 population and was considered putatively linked to the HtN gene for leaf blight resistance. This study shows that the RAPD technique does have application in maize breeding programmes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

The eldana stalk borer (Eldana saccharina Walker) is the most serious pest of the Southern African sugarcane industry, and it is imperative that effective control measures are available to minimize economic damage. Because conventional control methods have had limited success, cultivar resistance is seen as the most viable method of controlling infestation. However, due to the space- and time-consuming nature of the present screening methods, only small numbers of cultivars can be tested relatively late in the Plant Breeding selection programme. Increased resistance in breeding and selection populations is therefore slow. Buds are a preferred entry point of eldana larvae as they are softer than the rind that is present on the rest of the stalk surface. Preliminary results by other workers suggested that near infrared spectroscopy (NIRS) could provide a rapid screening method for the chemical profile in bud scales, the outer coating of buds and therefore the first contact point of an invading larva. If feasible, analysis of samples using this method could be done in the South African Sugar Experiment Station's (SASEX) stage two selection trials, providing an early indication of eldana resistance on large numbers of cultivars, without the necessity of separate trials. However, knowledge of how environments, position of bud scales on the stalk and age affect NIRS is required in order to determine the feasibility of the method. Planting of a trial with an identical set of genotypes across a range of environments, sampled at a number of ages, would provide the necessary information on environmental effects, whilst simultaneously providing the necessary range of samples to develop a calibration between bud scale chemical profiles and eldana resistance ratings. Inheritance patterns of the characteristics being measured is also required if they are to be used in a breeding programme. The original work by Rutherford (1993) was carried out on only five calibration sets (a set of standard clones with relatively well-known eldana resistance ratings), and different sets were not comparable due to what was assumed to be environmental differences between calibration sets. One aspect of the current experiment was to examine more closely the effect of genotype x environment interaction (G x E) on the performance of the NIRS technique under a range of conditions. Two sites were chosen to represent the conditions encountered in trials carried out by SASEX. The crops were sampled at three ages, representing the range of ages at which sugarcane is harvested in South Africa. Two locations on the stalk were also examined, top and bottom, for removal of bud scales, based on the assumption that aging of bud scales may affect chemical composition. A new NIRSystems 6500 instrument was acquired during the course of this study. Data from the new instrument indicated that there were no longer differences between the different calibration sets, and therefore no longer differences between environments. Spectra for different samples were very close, the differences being of the same scale as those recorded with repeated measures of the same samples, or between the readings for the standard solvent solution. This led to the conclusion that the differences observed on the original NIRSystems 5000 instrument were due to instrument error, not environmental differences. More importantly, the different calibration sets were not comparable despite being similar to each other. Prediction from one calibration set to another was low. These observations led to the conclusion that NIRS was not a suitable method for determining chemical compounds associated with tolerance of sugarcane genotypes to eldana borer. The original NIRS instrument was subject to error, and the small number of calibration sets included in the study led to the erroneous conclusion that NIRS was suitable for the prediction of varietal tolerance to eldana. With the acquisition of the new instrument, the errors generated by the old instrument became apparent. With the increase in number of calibration sets included in the study, it also became apparent that a global calibration covering all environments was not possible. An analysis of the heritability of the chemical compounds associated with eldana resistance was also included in this study. A biparental progeny design of 24 crosses with 33 unselected offspring per cross was used. This trial would have been analysed once the calibration had been developed using the environmental trial, and it would have provided knowledge of the breeding behaviour of the chemical compounds associated with tolerance to eldana. Because the NIRS technique proved to be unsuitable for detection of chemical compounds associated with eldana resistance, the heritability of these chemical compounds could not be studied. As the NIRS study did not produce data, the G x E interaction analysis and determination of heritability was applied to the bud scale mass data set. This study showed a relatively low positive correlation between bud scale mass and resistance to eldana. The broad sense heritability estimate for bud scale mass from the G x E interaction analysis was 0.45, and the narrow sense heritability estimate from parent-offspring regression analysis was approximately 0.27, suggesting a low degree of genetic determination in bud scale mass. The G x E interaction analyses gave varying results depending on the method used. The ANOVA analysis suggested that ages, sites and years had an effect on bud scale mass, while deviation from maximum plot showed no significance for G x E interactions. The number and choice of genotypes selected as unstable also varied with the method used to determine the stability of individual genotypes. Regression analysis and rank order analysis revealed a number of unstable genotypes, whilst stability variance and ecovalence, which produced similar results, detected only two unstable genotypes. In the rank order analysis correction of data to remove genotype effect, reduced the number of unstable genotypes, suggesting that the G x E interaction effect was partially confounded with the bud scale mass of the genotypes. This was a more reliable method than the uncorrected rank order analysis, and would be the preferred analysis type of all those tried. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.