Thesis (doctoral)--Lund University, 1995.

Thesis (doctoral)--Lund University, 1995.

Philosophiae Doctor - PhD / Promoter detection, especially in prokaryotes, has always been an uphill task and may remain so, because of the many varieties of sigma factors employed by various organisms in transcription. The situation is made more complex by the fact, that any seemingly unimportant sequence segment may be turned into a promoter sequence by an activator or repressor (if the actual promoter sequence is made unavailable). Nevertheless, a computational approach to promoter detection has to be performed due to number of reasons. The obvious that comes to mind is the long and tedious process involved in elucidating promoters in the ‘wet’ laboratories not to mention the financial aspect of such endeavors. Promoter detection/prediction of an organism with few characterized promoters (M.tuberculosis) as envisaged at the beginning of this work was never going to be easy. Even for the few known Mycobacterial promoters, most of the respective sigma factors associated with their transcription were not known. If the information (promoter-sigma) were available, the research would have been focused on categorizing the promoters according to sigma factors and training the methods on the respective categories. That is assuming that, there would be enough training data for the respective categories. Most promoter detection/prediction studies have been carried out on E.coli because of the availability of a number of experimentally characterized promoters (+- 310). Even then, no researcher to date has extended the research to the entire E.coli genome. / South Africa

Indiana University-Purdue University Indianapolis (IUPUI) / Cyclin Dependent Kinase regulation is often manipulated by cancer cells to promote unlimited proliferation. P27 is an important regulator of Cyclin E/CDK 2, which has been found in low amounts in many types of malignant cancers. Lovastatin has been shown to cause cell cycle arrest in the G1 phase of the cell cycle by increasing the P27 protein. There has been some question, however, if lovastatin regulates P27 at the transcriptional or translational level. Although it has been claimed that P27 expression regulation is due to an IRES located in its 5’UTR, other studies suggested that P27 expression is regulated at the level of transcription. To further investigate the regulation mechanism of P27 expression, the 5’-UTR of P27 and its deletion mutants were examined using a luciferase reporter gene in HeLa cells following exposure to lovastatin. It was found that lovastatin stimulated a significant 1.4 fold increase in its promoter activity of the full length 5’UTR (575). Deletion of 35 nucleotides from the 5’ end of the UTR eliminated the lovastatin-induced increase in promoter activity. Further mapping analyses of the first 35 bases showed that two regions, M1 (575-559) and M3 (543-527), were less sensitive to lovastatin than the other mutated constructs. Since M1 and M3 still showed some activity, a construct was created with deletions in both the M1 and M3 regions. This showed no increase in luciferase activity when exposed to lovastatin. Looking at RNA levels, there was a 1.5 fold increase in RNA when the full length 5’UTR was inserted into HeLa cells and exposed to 81 µM of lovastatin. In contrast, there was no increase in RNA when M1/M3 (575-559; 543-527) was inserted into HeLa cells and exposed to 81 µM of lovastatin. In addition, there was a 1.6 fold increase in endogenous P27 RNA levels after HeLa cells were exposed to 81 µM of lovastatin. In all of these experiments, there seems to be two promoters that work cooperatively: M1 (575-559) and M3 (543-527).

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”.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Understanding the complex nature of grapevine molecular biology is of great importance for viticulturists. Progress in the elucidation of key events on a genetic level could provide further insight into the underlying cues responsible for the precise control of physiological and metabolic changes during a specific condition such as fruit development. The use and analysis of molecular ‘tools’, such as promoters controlling the site and level of gene activity, could assist in the understanding of grapevine biology and serve as a platform for the future design and development of recombinant DNA protocols and strategies for Vitis vinifera L. A high-throughput gene expression system, cDNA-AFLPs, was successfully used to analyse large-scale transcriptional activity during berry ripening. Candidate cDNA fragments were selected on the basis of desired expression patterns and/or known gene function for subsequent promoter isolation. From three candidate cDNAs selected, the promoter of a gene encoding vacuolar pyrophosphatase (V-PPase) was isolated for computational and comparative analyses. Promoter activity was evaluated on a transient level using the green fluorescent protein (GFP) reporter gene. Comparative integration has allowed for putative correlation of cis-elements, acting as receptors within promoter regions, to regulate V-PPase gene expression in response to development, environmental stress and tissue-specificity. In this study, integration of genetic data have advanced the understanding and transcriptional role of a key enzyme (V-PPase) during grape ripening. Although never a replacement for experimental verification, this integrative strategy of combining gene expression profiles with bioinformatics and regulatory data will greatly assist in further elucidation of various other key components and regulatory cues associated with grapevine molecular biology. This study has allowed us to use molecular tools that could assist in gaining further insight into genetic complexities and could serve as a platform for a more refined genetic manipulation strategy in Vitis vinifera L. / AFRIKAANSE OPSOMMING: Begrip van die komplekse aard van wingerd molekulêre biologie is van groot belang vir wingerdkundiges. Vooruitgang in die begrip van belangrike gebeurtenisse op ń genetiese vlak behoort verdere insig in die onderliggende instruksies vir die noukeurige beheer van fisiologiese en metaboliese veranderinge tydens ń spesifieke kondisie soos vrug rypwording te bevorder. Die gebruik en analise van molekulêre ‘instrumente’ soos promoters, wat die posisie en vlak van geen aktiwiteit beheer, kan bydra tot n beter begrip van wingerd biologie en sodoende dien as ń platform vir die toekomstige ontwerp en ontwikkeling van rekombinante DNS (deoksiribonukleiensuur) protokolle en strategieë vir Vitis vinifera L. ń Hoë-kapasiteit geen uitdrukkings sisteem, nl. kDNS-AFLPs (komplementêre deoksiribonukleiensuur- geamplifiseerde fragment lengte polimorfisme), is suksesvol gebruik vir die analise van grootskaalse transkripsionele aktiwiteit tydens druif rypwording. Kandidaat kDNS fragmente is geselekteer, gebaseer op verlangde uitdrukkings-patrone en/of bekende geen funksie vir daaropvolgende promoter isolering. Van drie geselekteerde kandidaat kDNS fragmente, is die promoter van ń geen wat vakuolêre pirofosfatase (V-PPase) kodeer geïsoleer vir rekenaar- en vergelykende analise. Promoter aktiwiteit is op ń nie-stabiele vlak deur die gebruik van ń groen-fluoresserende proteien (GFP) verklikker geen geëvalueer. Vergelykende integrering het dit moontlik gemaak om veronderstelde korrelasies van cis-elemente, wat as reseptore binne ń promoter area dien, en die regulering van V-PPase geen uitdrukking, in reaksie tot ontwikkeling, omgewings stres en weefsel-spesifisiteit, te maak. Tydens hierdie studie, het die integrering van genetiese data gehelp om die transkripsionele rol van ń belangrike ensiem (V-PPase) tydens druif rypwording beter te verstaan. Alhoewel dit nooit ń plaasvervanger vir eksperimentele bewyse sal wees nie, kan hierdie gëintegreerde strategie, wat die kombinasie van geen-uitdrukkingsprofiele met bioinformatika en regulatoriese data behels, grootliks bydra om verskeie ander belangrike komponente en regulatorieseaanwysings geassosieërd met wingerd molekulêre biologie te ontrafel. Hierdie studie het verdere insig in genetiese kompleksiteite verleen, en kan nou dien as ń platform vir ń meer presiese genetiese manipulering strategie in Vitis vinifera L.

Chan Ka Ping Sophie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 140-144). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Acknowledgements --- p.ii / Abstract --- p.viii / 摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xii / List of Tables --- p.xiv / Chapter 1 --- Introduction / Chapter 1.1 --- Pathogenesis of HBV Infection --- p.1 / Chapter 1.2 --- Classification and Structure --- p.2 / Chapter 1.3 --- HBV Genome --- p.4 / Chapter 1.4 --- Replication Cycle --- p.7 / Chapter 1.5 --- HBV Genotypes and Nomenclature --- p.9 / Chapter 1.5.1 --- Asian prevalent genotypes --- p.9 / Chapter 1.5.2 --- Numbering system --- p.9 / Chapter 1.6 --- Identification of Markers in HBV Genome for HCC Development --- p.11 / Chapter 1.7 --- Project Objective --- p.13 / Chapter 1.8 --- Promoters of HBV --- p.14 / Chapter 1.8.1 --- Pre-S1 promoter --- p.14 / Chapter 1.8.2 --- X promoter and enhancer I --- p.14 / Chapter 1.8.3 --- Core promoter and enhancer II --- p.15 / Chapter 1.8.4 --- Pair of mutations at BCP --- p.17 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Construction of pGL3-promoter Plasmids --- p.18 / Chapter 2.1.1 --- Templates selection --- p.18 / Chapter 2.1.2 --- Amplification of promoters --- p.19 / Chapter 2.1.3 --- Cloning into pGL3-basic vector --- p.21 / Chapter 2.1.4 --- Screening and plasmid preparation --- p.21 / Chapter 2.2 --- Construction of Mutant Promoter Clones --- p.23 / Chapter 2.2.1 --- Site-directed mutagenesis --- p.23 / Chapter 2.2.2 --- pPreS 1 /2712C mutant clone --- p.24 / Chapter 2.3 --- Cloning of Full-length HBV Genomes --- p.26 / Chapter 2.3.1 --- Replication-competent HBV clone --- p.26 / Chapter 2.3.2 --- Amplification of full-length HBV genome --- p.28 / Chapter 2.3.3 --- Cloning into pUC19 vector --- p.28 / Chapter 2.3.4 --- Screening for insert and sequence confirmation --- p.29 / Chapter 2.3.5 --- Excision of full-length HBV from plasmid --- p.29 / Chapter 2.4 --- Re-construction into a 1.3-fold HBV Clone --- p.32 / Chapter 2.4.1 --- Cloning of HBV fragment nucleotide 979-2617 (nt 979-2617) --- p.32 / Chapter 2.4.2 --- Screening for insert and sequence confirmation --- p.33 / Chapter 2.4.3 --- Cloning of HBV fragment (nt 905-2000) --- p.33 / Chapter 2.4.4 --- Construction of a 1.3-fold HBV genotype Cs clone --- p.34 / Chapter 2.5 --- Cell Culture --- p.37 / Chapter 2.5.1 --- Cell culture maintenance --- p.37 / Chapter 2.5.2 --- Transient transfection of promoter clones --- p.37 / Chapter 2.5.3 --- Transient transfection of HBV genomes --- p.38 / Chapter 2.6 --- Dual-Luciferase® Reporter Assay System --- p.40 / Chapter 2.6.1 --- Principle of the assay --- p.40 / Chapter 2.6.2 --- Cell harvest --- p.43 / Chapter 2.6.3 --- Luciferase assay --- p.43 / Chapter 2.7 --- Data Analysis --- p.44 / Chapter 2.8 --- Extraction of HBV DNA from Intracellular Cores --- p.45 / Chapter 2.8.1 --- Harvest of intracellular cores --- p.45 / Chapter 2.8.2 --- Phenol/chloroform extraction --- p.45 / Chapter 2.9 --- Southern Blotting --- p.47 / Chapter 2.9.1 --- Transfer of DNA to membrane --- p.47 / Chapter 2.9.2 --- Preparation of probes --- p.47 / Chapter 2.9.3 --- Hybridization with radiolabeled probes --- p.48 / Chapter 2.10 --- Detection of HBeAg and HBsAg --- p.50 / Chapter 2.10.1 --- HBsAg assays --- p.50 / Chapter 2.10.2 --- HBeAg assays --- p.51 / Chapter 2.11 --- SEAP Reporter Gene Assay --- p.52 / Chapter 3 --- Results / Chapter 3.1 --- Templates Selected --- p.53 / Chapter 3.2 --- Results of Luciferase Assays --- p.58 / Chapter 3.2.1. --- BCP mutation of genotype A as control --- p.58 / Chapter 3.2.2. --- Effect of C1165T mutation on Xpro/enhI activity of HBV genotype B --- p.60 / Chapter 3.2.3. --- Effect ofT2712C mutation on pre-S1 promoter activity of HBV Genotype B --- p.60 / Chapter 3.2.4. --- Effect of G1613A mutation on core pro/enhII activity of HBV Genotype Cs --- p.64 / Chapter 3.2.5. --- G1613A and BCP mutation --- p.67 / Chapter 3.3 --- Full-length HBV Genome Clones --- p.70 / Chapter 3.3.1. --- Construction of replication-competent full-length HBV genome clones --- p.70 / Chapter 3.3.2. --- Drawbacks of the system --- p.78 / Chapter 3.4 --- Construction of a Replication-competent 1.3-fold HBV Clone --- p.82 / Chapter 3.4.1. --- Construction of the HBV (nt 979-2617) clone --- p.82 / Chapter 3.4.2. --- Construction of the HBV (nt 905-2000) clone --- p.86 / Chapter 3.4.3. --- Construction of 1.3-fold genotype Cs HB V clone --- p.89 / Chapter 3.4.4. --- Test for replication competency --- p.92 / Chapter 4 --- Discussion / Chapter 4.1 --- BCP Mutation as Control of the Luciferase Assay --- p.94 / Chapter 4.2 --- Promoter Activities Not Altered by T2712C and C1165T --- p.96 / Chapter 4.3 --- Mutation G1613A of Core pro/enhll --- p.98 / Chapter 4.3.1 --- Mutation resides in negative regulatory element of core promoter --- p.98 / Chapter 4.3.2 --- NRE and NRE-binding protein --- p.98 / Chapter 4.3.3 --- Relationship with BCP mutation --- p.101 / Chapter 4.4 --- HBV Constructs --- p.103 / Chapter 4.4.1 --- Rationale in re-construction of 1.3-fold HB V clone --- p.103 / Chapter 4.4.2 --- Replication competency --- p.104 / Chapter 4.5 --- Conclusion --- p.106 / Chapter 4.6 --- Future Work --- p.107 / Appendix --- p.108 / References --- p.140

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 179-198). Also issued on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 135-149). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 133-147). Also available on the Internet.