Indiana University-Purdue University Indianapolis (IUPUI) / Transcription and alternative splicing play important roles in HIV-1 gene expression and replication and mandate complicated but coordinated interactions between the host and the virus. Studies from our group have shown that a HIV-1 Tat-interacting protein of 110 kDa, Tip110 synergies with Tat in Tat-mediated HIV-1 gene transcription and replication. However, the underlying molecular mechanisms were not fully understood and are the focus of the dissertation research. In the study, we first demonstrated that Tip110 bound to unphosphorylated RNA polymerase II (RNAPII) in a direct and specific manner. We then showed that Tip110 was detected at the HIV-1 long terminal repeat (LTR) promoter and associated with increased phosphorylation of serine 2 within the RNAPII C-terminal domain (CTD) and increased recruitment of positive transcription elongation factor b (P-TEFb) to the LTR promoter. Consistent with these findings, we demonstrated that Tip110 interaction with Tat directly enhanced transcription elongation of the LTR promoter. During these studies, we also found that Tip110 altered HIV-1 mRNA alternative splicing and increased tat mRNA production. Subsequent analysis indicated that Tip110 selectively increased tat exons 1-2 splicing by activating HIV-1 A3 splice site but had no function in tat exons 2-3 splicing. We then showed that the preferential splicing activity of Tip110 resulted from Tip110 complex formation with hnRNP A1 protein, a negative splicing regulator that binds to the ESS2 element within tat exon 2, and as a result, blocked the complex formation of hnRNP A1 with ESS2 and subsequently activated HIV-1 A3 splice site. Taken together, these results show that Tip110 functions to regulate HIV-1 transcription elongation and HIV-1 RNA alternative splicing. These findings not only add to our understanding of Tip110 biology and function but also uncover a new potential target for development of anti-HIV intervention and therapeutic strategies.

The mRNA coding for the ammonium inducible NADP-specific glutamate dehydrogenase (NADP-GDH) from Chlorella was studied in induced and uninduced cells to determine the molecular mechanisms which regulate the cellular levels of this enzyme. A procedure for isolation of a high yield of total undegraded cellular polysomes was developed. The crosslinking reagent, dimethyl suberimidate, was employed to prepare a stable NADP-GDH-crosslinked-Sepharose-4B antigen affinity column for the purification of rabbit anti-NADP-GDH IgG. Binding studies with ¹²⁵I-labelled antibody and total polysomes, isolated from induced and uninduced cells, showed that the NADP-GDH was being synthesized on polysomes from both types of cells. When poly(A)- containing RNA was extracted from polysomes isolated from induced and uninduced cells, and translated in an mRNA-dependent in vitro translation system, NADP-GDH antigen was synthesized from the RNA from both sources. Based on sucrose density gradient analysis, Chlorella NADP-GDH mRNA has a sedimentation coefficient of 18 Comparison of the amounts of NADP-GDH synthesized in vitro from poly(A)-containing RNA and non-poly(A)-containing RNA showed the NADP-GDH mRNA contained polyadenylic acid sequence. By use of an indirect immunoadsorption procedure, the NADP-GDH mRNA was purified five- to sevenfold from total poly(A)-containing RNA. The overall purification of the NADP-GDH mRNA from total polysomal RNA was approximately two hundred-fold. Complementary DNA was synthesized from the partially purified RNA with reverse transcriptase. The cDNA sequences hybridized to the least abundant class of mRNA sequences present in total poly(A)-containing RNA. In vitro translation of total poly(A)-containing RNA showed that NADP-GDH synthesis was 0.1% of total protein synthesis. Upon addition of inducer to previously uninduced, synchronous cells, the amount of translatable NADP-GDH mRNA increased in a linear fashion after 30 min of the induction period. A change in rate of NADP-GDH mRNA accumulation was observed after 30 min of the induction period. The results support the prediction that since the NADP-GDH enzyme is unstable in vivo, during periods of NADP-GDH accumulation, the NADP-GDH mRNA accumulates. When poly(A)-containing RNA, isolated from uninduced synchronous cells was translated in vitro, NADP-GDH antigen was synthesized at each time in the cell cycle examined. The amount of translatable NADP-GDH mRNA increased throughout the cell cycle with a rate change occuring during the S-phase. This pattern of NADP-GDH mRNA accumulation is consistent with the hypothesis that NADP-GDH mRNA accumulates in uninduced cells at a rate proportional to gene dosage. These results provide one explanation for the observed pattern of enzyme potential in synchronous cells cultured in the absence of inducer. The data are consistent with the possibility that a single mRNA, which is subject to post-transcriptional modification by the inducer, codes for NADP-GDH. / Ph. D.

Face à un environnement changeant, la cellule a dû développer des systèmes de régulation lui permettant de s’adapter et d’assurer son développement et sa survie. Ces systèmes de régulation s’organisent autour de réseaux de régulation transcriptionnelle permettant l’expression des gènes codant pour les protéines dont la cellule a besoin. Dans la plupart des réseaux trancriptionnels, la régulation de la transcription des gènes est « raffinée » par la présence de circuits de rétroaction positifs et négatifs à l’origine de deux types de comportements différents: la multistabilité d’une part, et les comportements homéostatiques ou oscillants d’autre part. Deux réseaux de régulation transcriptionnelle de complexité différente ont été étudiés au cours de cette thèse :le réseau p53-Mdm2 impliqué dans l'arrêt de la croissance cellulaire, la réparation de l’ADN et l’apoptose chez les mammifères, et le réseau de facteurs transcriptionnels GATA impliqué dans la régulation du catabolisme de l’azote chez la levure Saccharomyces cerevisiae. L’analyse théorique du réseau p53-Mdm2 a eu pour principal objectif de reproduire et d’interpréter les données expérimentales disponibles dans la littérature concernant la réponse oscillante de la p53 lorsque l’ADN de la cellule est endommagé. L’analyse théorique des comportements du réseau GATA, quant à elle, a été couplée à une étude expérimentale dans les milieux de qualité intermédiaire en azote peu investigués jusqu’à présent. Pour analyser les propriétés dynamiques de ces deux réseaux, plusieurs approches complémentaires, se situant à différents niveaux de description, ont été utilisées: l’approche logique, différentielle et stochastique.<p>La première partie de cette thèse a été consacrée à l’étude du réseau p53-Mdm2 pour lequel nous avons développé un modèle simple composé d’un circuit de rétroaction positif imbriqué dans un circuit de rétroaction négatif. Les résultats de notre analyse logique montrent que les principales propriétés dynamiques du réseau peuvent être résumées par un petit nombre de diagrammes de bifurcation logique. Ces scénarios de bifurcations diffèrent par la séquence d’activation du circuit positif et négatif composant le réseau et dépendent d’une part de l’affinité de la p53 pour ses gènes cibles et d’autre part de son activité transcriptionnelle. Nous proposons que différents stress et types cellulaires pourraient correspondre à différents scénarios de bifurcation et donc conduire à des réponses différentes après irradiation. Cette première analyse qualitative nous a permis de rendre compte de différents aspects de la dynamique du réseau observés expérimentalement, tels que le changement de fréquence des oscillations en cours de réponse, les oscillations de longue durée de la p53 ou l’amortissement rapide des oscillations à l’échelle d’une population de cellules. Pour nous affranchir des fortes non-linéarités inhérentes au traitement logique, nous avons ensuite traduit le modèle logique en un modèle différentiel et montré que les principaux comportements présentés par le modèle logique sont conservés, suggérant que la structure du réseau détermine dans une large mesure les principales potentialités dynamiques du système. L’analyse des propriétés de bifurcation du modèle différentiel en fonction du niveau de dommage à l’ADN nous a également permis de mettre en évidence la présence de deux régimes oscillants d’amplitude, de valeur moyenne et de fréquence nettement différentes, séparés par une zone de bicyclicité où ces deux régimes coexistent. Cette propriété permet d’expliquer l’existence des deux fréquences d’oscillation différentes qui ont été observées expérimentalement en fonction de la dose d’irradiation. Enfin l’analyse stochastique de notre modèle nous a, en particulier, permis de rendre compte de l’augmentation du nombre de cellules oscillant à des fréquences élevées lorsque la dose d’irradiation augmente, observée expérimentalement.<p>La deuxième partie de notre thèse a été consacrée à l’étude du réseau de facteurs GATA chez la levure S.cerevisiae. Ce réseau, constitué des activateurs Gln3 et Nil1 et des répresseurs Dal80 et Gzf3, comporte plusieurs circuits de rétroaction positifs et négatifs interconnectés. Dans le but d’aider à comprendre le rôle et le fonctionnement du réseau GATA, nous avons effectué une analyse théorique et expérimentale de son comportement dynamique en fonction de la qualité de la source azotée. L’analyse différentielle montre la possibilité d’un comportement bistable dans les milieux de qualité intermédiaire en azote et d’oscillations amorties suite à un transfert nutritionnel d’une condition azotée à une autre, lorsque l’activation des gènes du réseau par Gln3 et Nil1 est synergique ou lorsque le gène Gln3 est supprimé. Gzf3 serait le répresseur clef impliqué dans la bistabilité tandis que Dal80 serait le répresseur clef impliqué dans les comportements oscillants. L’analyse stochastique nous a permis d’étudier l’effet des fluctuations moléculaires sur ces comportements et les distributions de variables importantes du système dans une population de cellules. Pour le modèle synergique de la souche sauvage et celui du mutant gln3°, elle a montré l’existence, dans des milieux de qualité intermédiaire en azote, de deux populations de cellules qui coexistent :une population où l’expression de Dal80 est réprimée, une autre où son expression est activée. Enfin, l’étude de la dynamique du couplage entre la protéine fluorescente Gfp, sous le contrôle du promoteur de DAL80, et le réseau GATA montre que, pour des ordres de grandeur physiologique de la vitesse de disparition de la Gfp, la bimodalité présente au niveau du réseau GATA devrait se refléter au niveau de la Gfp.<p>Les comportements bistables et oscillants mis en évidence dans notre étude théorique du réseau GATA ont ensuite été testés expérimentalement en suivant la Gfp sous le contrôle du promoteur de DAL80 en fonction de la concentration de la source azotée glutamine. Cette étude expérimentale nous a permis de mettre en évidence l’existence d’oscillations amorties de la fluorescence de la protéine de fusion Dal80-Gfp. De telles oscillations cependant n’ont pas été observées dans les expériences réalisées sur les autres souches testées pour lesquelles le gène de la Gfp est fusionné au promoteur de DAL80. Notre étude expérimentale montre également l’existence, chez la souche sauvage, d’une population unique de cellules fluorescentes quelle que soit la concentration du milieu extérieur en glutamine testé (0.2mM à 10mM). Un modèle additif où l’activation des gènes du réseau par Gln3 et Nil1 n’est pas synergique serait donc en meilleur accord avec nos observations. Chez les souches où le facteur Gln3 est inactivé, par contre, deux populations cellulaires, l’une de forte fluorescence et constituée de cellules de grande taille, l’autre de plus faible fluorescence et constituée de cellules de taille plus petite, coexistent pour des concentrations intermédiaires du milieu extérieur en glutamine. La forte corrélation observée entre la taille et la fluorescence des cellules suggère que le comportement bimodal observé au niveau de la fluorescence est lié au comportement bimodal observé au niveau de la taille. Enfin, un modèle phénoménologique de la croissance cellulaire nous a permis de reproduire l’existence de deux populations cellulaires de taille distincte.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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”.

The zinc finger associated domain (ZAD) family of transcription factors from Drosophila melanogaster is not well described in the literature, in part because it is very difficult to study by traditional mutagenesis screens. Bioinformatic studies indicate this is due to overlapping functions remaining after a recent evolutionary divergence. I set out to use in vitro-binding techniques to identify the characteristics of the ZAD family and test this theory. I have constructed glutathione S-transferase (GST)-ZAD domain chimeric proteins for use in pull down protein binding assays,and GST-Zinc finger (ZnF) array domain chimera for electrophoretic mobility shift assays (EMSA). Protein binding assays indicated two putative conserved interactors, similar to the analogous KRAB system in mammals. ... Competitive bindings were carried out to show a specificity of binding conferred by the identified conserved positions. While the consensus binding sites show relatively few similarities, the predicted target genes identified by the consensus binding sites show significant overlap. The nature of this overlap conforms to the known characteristics of the ZAD family but points to a more positive selection to maintain conservation of function. / by Joseph Krystel. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Mitochondrial disorders resulting from defects in oxidative phosphorylation are the most common form of inherited metabolic disease. Mutations in the human mitochondrial translation elongation factor GFM1 have recently been shown to cause the lethal pediatric disorder Combined Oxidative Phosphorylation Deficiency Syndrome (COXPD1). Children harboring mutations in GFM1 exhibit severe developmental, metabolic and neurological abnormalities. This work describes the identification and extensive characterization of the first known mutations in iconoclast (ico), the Drosophila orthologue of GFM1. Expression of human GFM1 can rescue ico null mutants, demonstrating functional conservation between the human and fly proteins. While point mutations in ico result in developmental defects and death during embryogenesis, animals null for ico survive until the second or third instar larval stage. These results indicate that in addition to loss-of-function consequences, point mutations in ico appear to produce toxic proteins with antimorphic or neomorphic effects. Consistent with this hypothesis, transgenic expression of a mutant ICO protein is lethal when expressed during development and inhibits growth when expressed in wing discs. In addition, animals with a single copy of an ico point mutation are more sensitive to acute hyperthermic or hypoxic stress. Removal of the positively-charged tail of the protein abolishes the toxic effects of mutant ICO, demonstrating that this domain is necessary for the harmful gain-of-function phenotypes observed in ico point mutants. / Further, expression of GFP-tagged constructs indicates that the C-terminal tail enhances ectopic nuclear localization of mutant ICO, suggesting that mislocalization of the protein may play a role in the antimorphic effects of mutant ICO. Taken together, these results illustrate that in addition to loss-of-function effects, gain-of-function effects can contribute significantly to the pathology caused by mutation in mitochondrial translation elongation factors. / by Catherine F. Trivigno. / Thesis (Ph.D.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

Longitudinals lacking gene (LOLA) is a transcription factor that is involved in a variety of axon guidance decisions in Drosophila melanogaster nervous system. Besides having a role as an epigenetic silencer and in the programmed cell death in Drosophila's ovary, this gene is also an example of complex transcription unit. LOLA is a transcription repressor and can generate 17 DNA - binding isoforms, through alternative splicing, each containing distinct zinc-finger proteins. This unique DNAbinding binding sequence to which LOLA-ZFP binds has been determined for four of the lola isoforms F, J, P and K. Also, bioinformatics' tool approach has been taken to identify the target genes that are regulated by these four LOLA splice variants. Future work will be done for the five other LOLA isoforms to categorize their putative DNA-binding sequences and subsequently their protein interactions. / by Bazila Qureshi. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

The assembly and maintenance of central synapses is a complex process, requiring myriad genes and their products. Highwire is a large gene containing a RING domain, characteristic of ubiquitin E3 ligases. Highwire has been shown to restrain axon growth and control synaptogenesis at a peripheral synapse. Here I examine the roles of Highwire at a central synapse in the adult Drosophila Giant Fiber System (GFS). Highwire is indeed necessary for proper axonal growth as well as synaptic transmission in the GFS. Differences arise between the central synapse and the neuromuscular junction (NMJ), where highwire was initially characterized : expresion from the postsynaptic cell can rescue highwire synaptic defects, which is not seen at the NMJ. In addition, a MAP kinase signaling pathway regulated by highwire at the NMJ has differing roles at a central synapse. Wallenda MAPK can rescue not only the highwire anatomical phenotype but also the defects seen in transmission. Another distinction is seen here : loss of function basket and Dfos enhance the highwire anatomical phenotype while expression of dominant negative basket and Dfos suppress the highwire phenotype. As a result we have compared the signaling pathway in flies and worms and found that the NMJ in the two organisms use a parallel pathway while the central synapse uses a distinct pathway. / by Kimberly Diane Rowland. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.

The zinc finger associated domain (ZAD) containing family of transcription factors is not well described in the literature, in part because it is very difficult to study by mutagenesis. We used in vitro-binding techniques to identify characteristics of the ZAD family, by constructing glutathione Stransferase (GST)-ZAD domain chimeric proteins for use in protein binding assays, and GST-Zinc finger array domain chimera for binding site selections. Protein binding assays indicated a possible shared cofactor, as seen in the analogous KRAB system in mammals. DNA binding assays have provided a consensus binding sequence for five of the ZAD proteins, consistent with previously reported work on ZAD and unpublished work on mammalian transcription factors. Research is ongoing with an additional ~50 ZAD proteins to more fully map the binding characters of ZAD proteins. / by Joseph Krystel. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Lee Wing Sum. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 206-226). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese Version) --- p.iv / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.3 / Chapter 2.1 --- Peroxisomes --- p.3 / Chapter 2.2 --- Peroxisome proliferators --- p.5 / Chapter 2.3 --- Human exposure pathways to peroxisome proliferators --- p.5 / Chapter 2.4 --- Peroxisome proliferator-induced pleiotropic effects in rodents --- p.7 / Chapter 2.4.1 --- Short-term effects --- p.7 / Chapter 2.4.1.1 --- Hepatomegaly --- p.7 / Chapter 2.4.2.1 --- Peroxisome proliferation --- p.8 / Chapter 2.4.1.3 --- Alteration of gene transcriptions --- p.8 / Chapter 2.4.2 --- Long-term effect --- p.9 / Chapter 2.5 --- Mechanisms of actions of peroxisome proliferators --- p.9 / Chapter 2.5.1 --- Substrate overload --- p.9 / Chapter 2.5.2 --- Receptor-mediated --- p.11 / Chapter 2.6 --- Peroxisome proliferator-activated receptors (PPARs) --- p.11 / Chapter 2.6.1 --- Structure of PPARs --- p.11 / Chapter 2.6.2 --- Tissue-specific expression of PPARs --- p.15 / Chapter 2.6.3 --- Physiological functions of PPARs --- p.19 / Chapter 2.6.3.1 --- PPARα --- p.19 / Chapter 2.6.3.2 --- PPARγ --- p.21 / Chapter 2.6.3.3 --- PPARδ --- p.23 / Chapter 2.7 --- Role of PPARα involved in peroxisome proliferator-induced pleiotropic responses --- p.24 / Chapter 2.7.1 --- Short-term effects --- p.24 / Chapter 2.7.2 --- Long-term effect --- p.24 / Chapter 2.8 --- Mechanisms of peroxisome proliferator-induced hepatocarcinogenesis --- p.25 / Chapter 2.8.1 --- Oxidative stress --- p.25 / Chapter 2.8.2 --- Suppression of apoptosis --- p.26 / Chapter 2.8.3 --- Increased cell proliferation --- p.27 / Chapter 2.9 --- Species difference to peroxisome proliferator-induced pleiotropic effects --- p.28 / Chapter 2.10 --- Fluorescent differential display (FDD) --- p.32 / Chapter Chapter 3 --- Objectives --- p.35 / Chapter Chapter 4 --- Materials and methods --- p.37 / Chapter 4.1 --- Animals and treatments --- p.37 / Chapter 4.1.1 --- Materials --- p.37 / Chapter 4.1.2 --- Methods --- p.37 / Chapter 4.2 --- Serum triglyceride and cholesterol analyses --- p.39 / Chapter 4.2.1 --- Materials --- p.41 / Chapter 4.2.2 --- Methods --- p.41 / Chapter 4.2.2.1 --- Serum preparation --- p.41 / Chapter 4.2.2.2 --- Triglyceride determination --- p.41 / Chapter 4.2.2.3 --- Cholesterol determination --- p.42 / Chapter 4.3 --- Statistical analysis --- p.42 / Chapter 4.4 --- Tail-genotyping --- p.42 / Chapter 4.4.1 --- Materials --- p.44 / Chapter 4.4.2 --- Methods. --- p.44 / Chapter 4.4.2.1 --- Preparation of genomic tail DNA --- p.44 / Chapter 4.4.2.2 --- PCR reaction --- p.45 / Chapter 4.5 --- Total RNA isolation --- p.45 / Chapter 4.5.1 --- Materials --- p.48 / Chapter 4.5.2 --- Methods --- p.48 / Chapter 4.6 --- DNase I treatment --- p.48 / Chapter 4.6.1 --- Materials --- p.49 / Chapter 4.6.2 --- Methods --- p.49 / Chapter 4.7 --- Reverse transcription of mRNA and fluorescent PCR amplification --- p.50 / Chapter 4.7.1 --- Materials --- p.50 / Chapter 4.7.2 --- Methods --- p.53 / Chapter 4.8 --- Fluorescent differential display (FDD) --- p.53 / Chapter 4.8.1 --- Materials --- p.53 / Chapter 4.8.2 --- Methods --- p.54 / Chapter 4.9 --- Excision of differentially expressed cDNA fragments --- p.54 / Chapter 4.9.1 --- Materials --- p.57 / Chapter 4.9.2 --- Methods --- p.57 / Chapter 4.10 --- Reamplification of differentially expressed fragments --- p.57 / Chapter 4.10.1 --- Materials --- p.60 / Chapter 4.10.2 --- Methods --- p.60 / Chapter 4.11 --- Subcloning of reamplified cDNA fragments --- p.62 / Chapter 4.11.1 --- PCR-TRAP® cloning system --- p.62 / Chapter 4.11.1.1 --- Materials --- p.63 / Chapter 4.11.1.2 --- Methods --- p.63 / Chapter 4.11.2 --- AdvaTage´ёØ PCR cloning system --- p.65 / Chapter 4.11.2.1 --- Materials --- p.65 / Chapter 4.11.2.2 --- Methods --- p.66 / Chapter 4.12 --- Purification of plasmid DNA from recombinant clones --- p.69 / Chapter 4.12.1 --- Materials --- p.69 / Chapter 4.12.2 --- Methods --- p.69 / Chapter 4.13 --- DNA sequencing of differentially expressed cDNA fragments --- p.70 / Chapter 4.13.1 --- CEQ 2000 Dye Terminator Cycle Sequence system --- p.71 / Chapter 4.13.1.1 --- Materials --- p.71 / Chapter 4.13.1.2 --- Methods --- p.71 / Chapter 4.13.2 --- ABI PRISM´ёØ dRhodamine Terminator Cycle Sequencing system --- p.72 / Chapter 4.13.2.1 --- Materials --- p.72 / Chapter 4.13.2.2 --- Methods --- p.72 / Chapter 4.13.3 --- Homology search against computer databases --- p.73 / Chapter 4.14 --- Northern analysis of differentially expressed cDNA fragments --- p.73 / Chapter 4.14.1 --- Formaldehyde gel electrophoresis of total RNA --- p.74 / Chapter 4.14.1.1 --- Materials --- p.74 / Chapter 4.14.1.2 --- Methods --- p.74 / Chapter 4.14.2 --- Preparation of cDNA probes for hybridization --- p.74 / Chapter 4.14.2.1 --- PCR DIG labeling --- p.75 / Chapter 4.14.2.1.1 --- Materials --- p.75 / Chapter 4.14.2.1.2 --- Methods --- p.75 / Chapter 4.14.2.2 --- Random Prime cDNA DIG labeling --- p.75 / Chapter 4.14.2.2.1 --- Materials --- p.75 / Chapter 4.14.2.2.2 --- Methods --- p.76 / Chapter 4.14.3 --- Purification of DNA from agarose gel --- p.77 / Chapter 4.14.3.1 --- Materials --- p.77 / Chapter 4.14.3.2 --- Methods --- p.78 / Chapter 4.14.4 --- Hybridization --- p.78 / Chapter 4.14.4.1 --- Materials --- p.78 / Chapter 4.14.4.2 --- Methods --- p.73 / Chapter 4.14.5 --- Synthesis of mouse GAPDH probe from normalization --- p.80 / Chapter 4.14.5.1 --- Materials --- p.80 / Chapter 4.14.5.2 --- Methods --- p.80 / Chapter Chapter 5 --- Results --- p.82 / Chapter 5.1 --- Liver morphology --- p.82 / Chapter 5.2 --- Liver weight --- p.82 / Chapter 5.3 --- Serum triglyceride and cholesterol levels --- p.88 / Chapter 5.4 --- Confirmation of genotypes --- p.91 / Chapter 5.5 --- DNase I treatment --- p.91 / Chapter 5.6 --- FDD RT-PCR and band excision --- p.98 / Chapter 5.7 --- Reamplification of excised cDNA fragments --- p.111 / Chapter 5.8 --- Subcloning of reamplified cDNA fragments --- p.121 / Chapter 5.9 --- DNA sequencing of subcloned cDNA fragments --- p.124 / Chapter 5.10 --- Confirmation of the differentially expressed cDNA fragments by Northern blot analysis --- p.132 / Chapter 5.11 --- Temporal expression pattern of differentially expressed genes --- p.157 / Chapter 5.12 --- Tissue distribution pattern of differentially expressed genes --- p.171 / Chapter Chapter 6 --- Discussions --- p.183 / Chapter 6.1 --- "Lack of hepatomegaly, hypotriglyceridemia and hepatic nodule formation in PPARα (-/-) mice" --- p.184 / Chapter 6.2 --- "Identification of PPARα-dependent and Wy-14,643 responsive genes" --- p.185 / Chapter 6.3 --- Functional roles of the isolated cDNA fragments --- p.186 / Chapter 6.3.1 --- Fragments B14 and H4 --- p.187 / Chapter 6.3.2 --- Fragment H1 --- p.189 / Chapter 6.3.3 --- Fragment H5 --- p.192 / Chapter 6.3.4 --- Fragment H8 --- p.194 / Chapter 6.4 --- Temporal expression patterns of the isolated cDNA fragments --- p.196 / Chapter 6.5 --- Tissue distribution patterns of the isolated cDNA fragments --- p.197 / Chapter Chapter 7 --- Conclusions --- p.200 / Chapter Chapter 8 --- Future studies --- p.204 / Chapter 8.1 --- Subcloning and characterization of the other differentially expressed genes --- p.204 / Chapter 8.2 --- Overexpression and inhibition expression of specific genes --- p.204 / Chapter 8.3 --- Generating transgenic mice with target disruption of specific gene --- p.205 / References --- p.206