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Crowther, Daniel. "Cloning and characterization of Cpf1P from Schizosaccharomyces pombe." Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.320634.
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Kiesler, Eva. "Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans." Doctoral thesis, Stockholm : Institutionen för molekylärbiologi och funktionsgenomik, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-218.
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Tang, Terry, and University of Lethbridge Faculty of Arts and Science. "Mathematical modeling of eukaryotic gene expression." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry, 2010, 2010. http://hdl.handle.net/10133/2567.
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Using the Gillespie algorithm, the export of the mRNA molecules from their transcription
site to the nuclear pore complex is simulated. The effect of various structures in the nu-
cleus on the efficiency of export is discussed. The results show that having some of the
space filled by chromatin near the mRNA synthesis site shortens the transport time. Next, the complete eukaryotic gene expression including transcription, splicing, mRNA export, translation, and mRNA degradation is modeled using delay stochastic simulation. This allows for the study of stochastic effects during the process and on the protein production rate patterns. Various protein production patterns can be produced by adjusting the poly-A tail length of the mRNA and the promoter efficiency of the gene. After that, the opposing effects of the chromatin density on the seeking time of the transcription factors for the promoter and the exit time of the mRNA product are discussed.xi, 102 leaves ; 28 cm
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Lahudkar, Shweta L. "REGULATION OF EUKARYOTIC GENE EXPRESSION BY mRNA CAP BINDING COMPLEX AND CAPPING MACHINERY." OpenSIUC, 2014. https://opensiuc.lib.siu.edu/dissertations/834.
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A characteristic feature of gene expression in eukaryotes is the addition of a 5' terminal 7-methylguanine "cap" to nascent pre-messenger RNA (mRNA) in the nucleus. It is the 5'capping process, which proves vital to creating a mature mRNA. The synthesis of an mRNA followed by its capping is a complex undertaking which requires a set of protein factors. The capped mRNA is then exclusively bound by a cap-binding complex (CBC). CBC shields mRNA from exonucleases as well as regulates downstream post-transcriptional events, translational initiation and nonsense mediated mRNA decay (NMD). Any misregulation during capping or in the binding of CBC can lead a number of diseases/disorders. Thus, the process and regulation of capping and CBC binding to mRNA are important fields to study the control of gene expression. Over the years, capping apparatus and CBC have been implicated in post-transcriptional regulation. However, it is not yet known whether CBC plays any role in controlling transcriptional initiation or elongation. Thus, the major research focus in my thesis had been to analyze the role of CBC and capping enzymes in regulation of transcriptional initiation and elongation. The results have revealed the role of CBC in stimulating the formation of pre-initiation complex (PIC) at the promoter in vivo via Mot1p (modifier of transcription). Subsequently, we have demonstrated the roles of CBC in transcription elongation, splicing and nuclear export of mRNA. Interestingly, we find that the capping enzyme, Cet1p, decreases promoter proximal accumulation of RNA polymerase II. These results support that Cet1p promotes the release of paused-RNA polymerase II to get engaged into elongating form for productive transcription. Such function of Cet1p appears to be mediated via the Facilitates chromatin transcription (FACT) complex. We find that FACT is targeted to the active gene by the N-terminal domain of Cet1p independently of its capping activity. In the absence of Cet1p, recruitment of FACT to the active gene is impaired, leading to paused-RNA polymerase II. Collectively, the results of my thesis work provide significant insight on the regulation of gene expression by CBC and capping enzyme, Cet1p.
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Waters, Lorna Catherine. "Solution structures of proteins and complexes involved in the regulation of eukaryotic gene expression." Thesis, University of Leicester, 2007. http://hdl.handle.net/2381/29722.
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The initial work focused on the interaction between the general coactivator CBP (SID domain) and members of the p160 family of coactivators (AD1 domain), which is a key step in the activation of transcription by nuclear receptors. The solution structure of the CBP SID / SRC1 AD1 complex described in this thesis shows that the two helical domains are intimately associated, with the first helix in SRC1 AD1 and the first three helices in CBP SID forming a four helix bundle, which is capped by the fourth helix of the AD 1 domain. Comparisons with the structure of the related CBP SID / ACTR AD 1 complex showed that while the CBP SID domain adopts a similar fold in complex with different p160 proteins, the topologies of the AD1 domains are strikingly different, a feature that is likely to contribute to functional specificity of these complexes. The second part of the work described here focused on the interaction between the MA-3 domains of the tumour suppressor Pdcd4 and the translation factor eIF4A, which has been shown to inhibit cap-dependent translation. The C-terminal MA-3 domain (Pdcd4 MA-3C) was shown to consist of three atypical HEAT repeats capped by a final helix. This domain was found to interact with the N-terminal domain of eIF4A through a conserved surface region. The comparison of NMR spectra obtained from Pdcd4 MA-3 C and the tandem MA-3 region strongly suggests that the tandem MA-3 region is composed of two equivalent domains connected by a semi-flexible linker. The high resolution structural information obtained provides important insights into the interactions and functional specificity of the protein complexes studied.
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Mehraein-Ghomi, Farideh. "Analysis of the assembly of a eukaryotic glucose transporter into the Escherichia coli cytoplasmic membrane." Thesis, University of Sussex, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.284076.
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Kiełbasa, Szymon M. "Bioinformatics of eukaryotic gene regulation." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2006. http://dx.doi.org/10.18452/15562.
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Die Aufklärung der Mechanismen zur Kontrolle der Genexpression ist eines der wichtigsten Probleme der modernen Molekularbiologie. Detaillierte experimentelle Untersuchungen sind enorm aufwändig aufgrund der komplexen und kombinatorischen Wechselbeziehungen der beteiligten Moleküle. Infolgedessen sind bioinformatische Methoden unverzichtbar. Diese Dissertation stellt drei Methoden vor, die die Vorhersage der regulatorischen Elementen der Gentranskription verbessern. Der erste Ansatz findet Bindungsstellen, die von den Transkriptionsfaktoren erkannt werden. Dieser sucht statistisch überrepräsentierte kurze Motive in einer Menge von Promotersequenzen und wird erfolgreich auf das Genom der Bäckerhefe angewandt. Die Analyse der Genregulation in höheren Eukaryoten benötigt jedoch fortgeschrittenere Techniken. In verschiedenen Datenbanken liegen Hunderte von Profilen vor, die von den Transkriptionsfaktoren erkannt werden. Die Ähnlichkeit zwischen ihnen resultiert in mehrfachen Vorhersagen einer einzigen Bindestelle, was im nachhinein korrigiert werden muss. Es wird eine Methode vorgestellt, die eine Möglichkeit zur Reduktion der Anzahl von Profilen bietet, indem sie die Ähnlichkeiten zwischen ihnen identifiziert. Die komplexe Natur der Wechselbeziehung zwischen den Transkriptionsfaktoren macht jedoch die Vorhersage von Bindestellen schwierig. Auch mit einer Verringerung der zu suchenden Profile sind die Resultate der Vorhersagen noch immer stark fehlerbehafted. Die Zuhilfenahme der unabhängigen Informationsressourcen reduziert die Häufigkeit der Falschprognosen. Die dritte beschriebene Methode schlägt einen neuen Ansatz vor, die die Gen-Anotation mit der Regulierung von multiplen Transkriptionsfaktoren und den von ihnen erkannten Bindestellen assoziiert. Der Nutzen dieser Methode wird anhand von verschiedenen wohlbekannten Sätzen von Transkriptionsfaktoren demonstriert.Understanding the mechanisms which control gene expression is one of the fundamental problems of molecular biology. Detailed experimental studies of regulation are laborious due to the complex and combinatorial nature of interactions among involved molecules. Therefore, computational techniques are used to suggest candidate mechanisms for further investigation. This thesis presents three methods improving the predictions of regulation of gene transcription. The first approach finds binding sites recognized by a transcription factor based on statistical over-representation of short motifs in a set of promoter sequences. A succesful application of this method to several gene families of yeast is shown. More advanced techniques are needed for the analysis of gene regulation in higher eukaryotes. Hundreds of profiles recognized by transcription factors are provided by libraries. Dependencies between them result in multiple predictions of the same binding sites which need later to be filtered out. The second method presented here offers a way to reduce the number of profiles by identifying similarities between them. Still, the complex nature of interaction between transcription factors makes reliable predictions of binding sites difficult. Exploiting independent sources of information reduces the false predictions rate. The third method proposes a novel approach associating gene annotations with regulation of multiple transcription factors and binding sites recognized by them. The utility of the method is demonstrated on several well-known sets of transcription factors. RNA interference provides a way of efficient down-regulation of gene expression. Difficulties in predicting efficient siRNA sequences motivated the development of a library containing siRNA sequences and related experimental details described in the literature. This library, presented in the last chapter, is publicly available at http://www.human-sirna-database.net
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Dörr, Katrin Zaragoza. "Molecular mechanisms of gene activation and gene expression mediated by CCAAT/enhancer binding proteins." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2008. http://dx.doi.org/10.18452/15873.
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Der Transkriptionsfaktor CCAAT/Enhancer-Binding Protein alpha (C/EBPa) koordiniert Proliferationshemmung und Differenzierung von myeloiden VorlŠuferzellen und Adipozyten. C/EBPa ist ein transkriptioneller Aktivator von abstammungspezifischen Genen und blockiert den Zellzyklus durch Repression von proliferationsfšrdernden E2F Zielgenen. Die hier gezeigten Daten zeigen, dass auch umgekehrt E2F die transkriptionelle und differenzierungsfšrdernde AktivitŠt von C/EBPa entgegenwirkt. Somit besitzen E2F-C/EBPa eine zentrale Schalterfunktion zwischen Proliferation und Differenzierung. Der Repressionsmechanismus durch E2F ist in mehreren Aspekten neuartig: Zum erstenmal wurde gezeigt, dass E2F einen anderen Transkriptionsfaktor reprimieren kann. E2F reprimiert die transkriptionelle AktivitŠt von C/EBPa ohne Bindung an cis-regulatorischen Elemente, sondern durch direkte Protein-Protein Interaktionen, die die Bindung von C/EBPa an DNA verhindern. Diese Form der transkriptionellen Repression geschieht unabhŠngig von "Pocket-Proteinen''". Patienten mit Akuter Myeloiden LeukŠmie (AML) weisen hŠufig eine gestšrte DNA Bindung von C/EBPa auf, welche ursachlich fŸr granulozitŠren Funktionsstšrungen sein kšnnte. Daher wŠre es wichtig zu analysieren ob E2F die DNA Bindung von C/EBPa in AML Patienten beeintrŠchtigt und ob auf E2F gerichtete Therapien granulozitŠre Reifung wiederherstellen. C/EBPa blockiert Zellproliferation durch vielseitigen Mechanismen. Hier wurde gezeigt, dass C/EBPa mit UBF1, dem Co-Aktivator der RNA Polymerase I, an chromosomalen Foci positioniert wird. Eine €hnlichkeit zu anderen fokalen Strukturen suggeriert, dass C/EBPa die Transkription von Polymerase I regulierten rRNA Gene reprimieren und somit ribosomale Biogenese beeintrŠchtigen kšnnte. Die Assoziation zwischen C/EBPa und UBF1 wird durch die Histon-Methyltransferase SUV39H1 stimuliert. Demnach kšnnte die antiproliferative Funktion von C/EBPa nicht nur auf der Regulierung von RNA Pol II-abhŠngiger Transkription, sondern auch auf der Repression von RNA Pol I regulierter rRNA Synthese basieren.The transcription factor CCAAT/Enhancer-Binding Protein alpha (C/EBPa) coordinates proliferation arrest and differentiation of myeloid progenitors and adipocytes. C/EBPa acts as a transcriptional activator of lineage specific genes and blocks the cell cycle by repressing transcription of E2F-regulated genes. Data presented here suggest that also inversely E2F interferes with the transcriptional activity of C/EBPa, counteracting C/EBPa-mediated differentiation processes. Thus, E2F-C/EBPa are part of a switch mechanism between proliferation and differentiation. The mechanism by which E2F suppresses C/EBPa-mediated transactivation is novel in several aspects. E2F acts as a co-repressor of another transcription factor, C/EBPa, without binding to cis-regulatory elements, but by direct protein-protein interactions that abolish the binding of C/EBPa to DNA. This mechanism of transcriptional repression occurs independent of pocket proteins. Disturbed DNA binding of C/EBPa is often observed in AML patients suggesting a causative role in granulocytic disorders. Thus, it would be of main interest to analyze whether E2F mediates disruption of C/EBPa''s DNA-binding in AML patients and whether therapies directed against E2F could restore granulocytic maturation. Despite the extensive knowledge of mechanisms involved in the inhibitory function of C/EBPa, it has not been addressed whether C/EBPa may impinge on cell proliferation by affecting the ribosomal biogenesis of a cell. This work demonstrates an association of C/EBPa to the RNA Pol I transcription factor UBF1, both proteins retained in large chromosomal foci. Similarities to other focal structures associated to UBF1, suggest that C/EBPa may repress transcription of Pol I-transcribed rRNA genes, and thus affect ribosomal biogenesis. The enrichment of C/EBPa at sites of UBF1 is induced by the histone methyltransferase SUV39H1. Thus, C/EBPa may not only control lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II, but also may act as a repressor of RNA Pol I mediated rRNA synthesis.
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Kiri, Arpna. "The isolation and function of the 3'untranslated region of the myosin heavy chain genes of skeletal muscle." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325611.
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Walton, Cherie. "Incorporation of Listeriolysin O into a ligand-based carrier system resulting in enhancement of gene expression /." Click for abstract, 1998. http://library.ctstateu.edu/ccsu%5Ftheses/1509.html.
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Thesis (M.A.)--Central Connecticut State University, 1998.Thesis advisor: Thomas King. "... in partial fulfillment of the requirements for the degree of Master of Arts in Biological Sciences." Includes bibliographical references (leaves 49-52).
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Roth, Robyn Lindsay. "The evaluation of heterologous eukaryotic expression systems for the production of biocatalytic enzymes." Thesis, Stellenbosch : Stellenbosch University, 2008. http://hdl.handle.net/10019.1/21733.
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Thesis (PhD)--Stellenbosch University, 2008.ENGLISH ABSTRACT: Heterologous gene expression is of considerable interest for the production of proteins of therapeutic and industrial importance. As the nature of recombinant proteins has become more complex and as transformation systems have been established in more species, so the variety of hosts available for expression has increased. Every system available has both advantages and disadvantages. The research presented here highlights the advantages of selecting the most appropriate expression system for different recombinant proteins. Expression of different biocatalytically-relevant enzymes, epoxide hydrolases, halohydrin dehalogenase, laccase and mannanase, in different host systems is undertaken, and expression levels and activity are compared. The development of Yarrowia lipolytica as a whole-cell biocatalyst is described. Y. lipolytica is used for the functional expression of epoxide hydrolases (EHs) and halohydrin dehalogenases. EHs are hydrolytic enzymes that convert epoxides to vicinal diols by ring-opening. Two new fungal EHs from Rhodosporidium toruloides NCYC 3181 and NCYC 3158 (a putative Cryptococcus curvatus strain) were identified and cloned. Additional EHs from different sources, including bacteria, yeasts, fungi and plants, were chosen for expression in Y. lipolytica, in order to determine its suitability as the expression system of choice for the production of EHs. Multi-copy integrants were developed, with the genes under control of the growthphase dependent hp4d promoter. A Saccharomyces cerevisiae strain was developed, expressing the EH from Rhodotorula araucariae1, to compare as a whole-cell biocatalyst with Y. lipolytica. This strain proved to be an exceptionally poor wholecell biocatalyst. All the Y. lipolytica strains developed showed varying levels of activity towards different classes of epoxides. Some strains displayed opposite enantioselectivities, allowing for potential complete conversions of racemic epoxides to the desired enantiomeric product. Halohydrins can be considered direct precursors of epoxides. Halohydrin dehalogenases catalyse the nucleophilic displacement of a halogen ion in halohydrins by a vicinal hydroxyl group, yielding an epoxide, a proton and a halide ion. The HheC gene from Agrobacterium radiobacter AD1, codon-optimised to match the codon usage of Y. lipolytica, was over-expressed in Y. lipolytica by generation of multi-copy integrants, further expanding the use of this organism as a host strain for heterologous production of enzymes. Expression levels were maximised by creating tandem repeats of the introduced HheC gene. The ring-closure activity with 2-chloro- 1-phenylethanol as substrate was demonstrated to be broadly dose-dependent. The b-mannanase gene (man1) from Aspergillus aculeatus MRC11624 was expressed in Y. lipolytica with effective secretion in the presence of its native secretion signal, using the hp4d promoter. The same gene was expressed in Aspergillus niger2 under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP) and the Aspergillus awamori glucoamylase terminator (glaT). Following optimisation with copy numbers and culture conditions, maximal activity levels of 26,140 nkat.ml-1 for Y. lipolytica, and 16,596 nkat.ml-1 for A. niger were obtained. Laccases are important enzymes for bioremediation, and the best characterised enzymes are from the fungus Trametes versicolor. The objective of this research was to optimise expression of T. versicolor laccases (lcc1 and lcc2) in A. niger D15 and Pichia pastoris3. The Lcc1 enzyme was less active than Lcc2 in both hosts. P. pastoris secreted 0.4 U.L-1 Lcc1 and 2.8 U.L-1 Lcc2, compared to 2,700 U.L-1 produced by A. niger. The Lcc2 enzyme from recombinant A. niger was subsequently purified and characterised in terms of molecular weight and glycosylation, and compared to the wild-type enzyme purified from T. versicolor. The work presented underscores the requirement for experimentation before finalising the choice of an expression system for the optimal production of the desired protein. Every system available has both advantages and disadvantages, and when considering which system to use for producing a recombinant protein, various factors must be taken into consideration. However, the choice is broad and each decision needs to be made empirically. 1 The construction of the S. cerevisiae epoxide hydrolase production strain was carried out by Dr Neeresh Rohitlall of CSIR Biosciences. The Y. lipolytica epoxide hydrolase strains were constructed by the author. 2 The construction of Man1-producing A. niger strain was done by Dr Shaunita Rose of the University of Stellenbosch. The construction of Y. lipolytica Man1 production strains was done by the author. 3 The expression of T. versicolor laccases in P. pastoris was done by Christina Bohlin of Karlstad University. A niger laccase production strains were created by the author.
AFRIKAANSE OPSOMMING: Heteroloë geen uitdrukking is van groot belang vir die produksie van proteïene wat van terapeutiese en industriele belang is. Soos die aard van rekombinante proteïene meer ingewikkeld raak en getransformasie-sisteme vir verskeie spesies gevestig raak, is daar ’n groter verskeidenheid van gashere beskikbaar vir geenuitdrukking. Elke sisteem het beide sy voor- en nadele. Hierdie navorsing beklemtoon die voordele wanneer die mees gepaste uitdrukkingssiteem gekies word. Die uitdrukking van verskeie ensieme van biokatalities belang, epoksiedhidrolases, halohidrien dehalogenase, lakkase en mannanase in verskillende gasheersisteme is onderneem en die uitdrukkingsvlakke en aktiwiteite vergelyk. Die ontwikkeling van Yarrowia lipolytica as ’n heelsel biokatalis word beskryf. Y. lipolytica word gebruik vir die funksionele uitdrukking van epoksiedhidrolases (EHs) en halohidrien dehalogenases. EHs is hidroliseringsensieme wat die epoksiede omskakel na aangrensende diole deur middel van ring-opening. Twee nuwe fungi EHs vanaf Rhodosporidiom toruloides NCYC 3181 en NCYC 3158 (’n moontlike Cryptococcus curvatus) is geïdentifiseer en gekloneer. Verdere EHs van verskillende bronne, insluitend bakterieë, giste, fungi en plante, is gekies vir uitdrukking in Y. lipolytica ten einde sy geskiktheid vir die produksie van EHs te bepaal. Multikopie integrante is ook ontwikkel met gene onder beheer van die groei-fase afhanklike hp4d promotor. ’n Saccharomyces cerevisiae ras is ook ontwikkel vir die uitdrukking van die EH van Rhodotorula araucariae4 sodat dit met Y. lipolytica as ’n heelsel biokatalis vergelyk kan word. Hierdie ras was ‘n buitengewone swak heelsel biokatalis. Al die Y. lipolytica rasse wat ontwikkel is het wisselende aktiwiteitsvlakke teenoor verskillende klasse van epoksiede getoon. Sommige rasse het teenoorgestelde enantio-selektiwiteit getoon en het die potensiaal om rasemiese epoksiede volledig na die gewensde enantiomeriese produk om te skakeling. Halohidriene kan as direkte voorgangers van epoksiede beskou word. Halohidrien dehalogenases kataliseer die nukleofiliese vervanging van ’n halogeen-ioon in halohidriene deur ’n aangrensende hidroksiel groep, wat ’n epoksied, ’n proton en ’n halied-ioon lewer. Die HheC geen van Agrobacterium radiobacter AD1 is kodon– geöptimiseer om te pas by die kodon gebruik van Y. lipolytica en was uitgedruk in Y. lipolytica deur die skep van mulitkopie integrante, ’n verdere verbreeding van die toepaslikheid van die organisme as gasheerras vir die heteroloë produksie van ensieme. Maksimum uitdrukkingsvlakke is bereik deur die skep van opeenvolgende herhalings van die ingevoegde HheC-geen. Daar is ook gewys dat die ring-sluitingsaktiwiteit met 2-chloro-1-pheniel-etanol as substraat meestal dosis-afhanklik is. Die -mannanase geen (man1) van Aspergillus aculeatus MRC11624 is uitgedruk en effektief in Y. lipolytica mbv sy eie uitskeidings sein uitgeskei, met die gebruik van die groei-fase afhanklike hp4d promotor. Dieselfde geen is uitgedruk in Aspergillus niger5 onder beheer van die A. niger gliseraldehied-3-fosfaat dehidrogenase promotor (gpdp) en die Aspergillus awamori glikoamilase termineerder (glaT). Verdere optimisering van kopiegetal en voedingskondisies het gelei tot maksimum aktiwiteitsvlakke van 26,140 nkat.ml-1 vir Y. lipolytica en 16,596 nkat.ml-1 vir A. niger. Lakkases is belangrike ensieme vir bio-remediëring, en die ensieme van die fungus Trametes versicolor is die beste gekarateriseer. Die doelwit van hierdie navorsing was die optimisering van die uitdrukking van T. versicolor lakkases (lcc1 en lcc2) in A. niger en Pichia pastoris6. Die Lcc1 ensiem was minder aktief as Lcc2 in altwee die gashere. P. pastoris het 0.4 U.L-1 Lcc1 en 2.8 U.L-1 Lcc2 onderskeidelik uitgeskei, in vergelyking met 2,700 U.L-1 Lcc2 wat deur A. niger geproduseer is. Die Lcc2 ensiem afkomstig van die rekombinante A. niger is vervolgens gesuiwer en gekarakteriseer met betrekking tot molekulêre massa en glikosilering, en daarna vergelyk met die wilde-tipe ensiem wat deur T. versicolor geproduseer word. Die werk wat hier aangebied word, beklemtoon die vereistes vir eksperimentering voor die finale keuse met betrekking tot ’n gepaste uitdrukkingsisteem gemaak kan word vir die optimale produksie van die gewensde proteïen. Elke sisteem het beide voordele en nadele, en wanneer ’n sisteem oorweeg word is daar verskeie faktore wat in ag geneem moet word. ’n wye verskeidenheid van keuses is beskikbaar en elke besluit moet empiries gemaak word. 4 Die konstruksie van die S. cerevisiae epoksiedhidrolase-produserende ras is deur Dr Neeresh Rohitlall van CSIR Biosciences gedoen. Die Y. lipolytica epoxied hydrolase rasse is deur die outeur gemaak. 5 Die konstruksie van die Man1-produserende A. niger ras is deur Dr Shaunita Rose van die Universiteit van Stellenbosch gemaak. Die Y. lipolytica Man1 ras is gemaak deur die outeur. 6 Die uitdrukking van T. versicolor lakkases in P. pastoris is gedoen deur Christina Bohlin van Karlstad University. Die A niger lakkase produksie ras is geskep deur die outeur.
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Oswald, Oliver. "Plastid redox state and sugars as interactive regulators of nuclear photosynthetic gene expression." Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368522.
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Wilhelm, Margareta. "The p53-induced gene wig-1 : regulation of expression and role in embryonic development /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-728-2.
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Li, Dajiang. "Studies on the expression of bFGF and IGF-I and -II in 3T3 cells." Thesis, University of the West of Scotland, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336899.
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Dellow, Kimberley Anne. "Identification and characterisation of factors binding the human cardiac troponin I gene promoter." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312168.
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Cogan, John G. "DNA-binding proteins regulating vascular smooth muscle alpha-actin gene expression in myoblasts and fibroblasts /." The Ohio State University, 1995. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487863429091115.
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Sever, Richard. "Roles of protein kinases in the regulation of AP-1 and associated transcription factors." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245165.
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Luo, Dan. "Novel cross-linking technologies to assess protein-DNA binding and DNA-DNA complexes for gene delivery and expression /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487946776022443.
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Ng, Ho-bun Dakilis. "Nutritional programming of hepatic IGF-1 expression in rats /." Hong Kong : University of Hong Kong, 2002. http://sunzi.lib.hku.hk/hkuto/record.jsp?B24709359.
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吳浩賓 and Ho-bun Dakilis Ng. "Nutritional programming of hepatic IGF-1 expression in rats." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B31226577.
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Giorgini, Flaviano. "Functional analysis of the murine sequence-specific RNA binding protein MSY4 /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/10293.
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Sukiennicki, Teresa Lyn. "Regulation of expression of the HLA class II gene, DQB1 /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/8358.
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Au, Y. K. Tiffany. "Investigating the molecular mechanisms of campomelic dysplasia in a mouse with a Sox9 gene mutation." Thesis, Click to view the E-thesis via HKUTO, 2007. http://sunzi.lib.hku.hk/HKUTO/record/B39557236.
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Tydén, Eva. "Cytochrome P450 3A and ABC-transport proteins in horse /." Uppsala : Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, 2008. http://epsilon.slu.se/200893.pdf.
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Adler, David A. "A tale of two x-linked genes : gene expression, localization and the Ohno hypothesis /." Thesis, Connect to this title online; UW restricted, 1996. http://hdl.handle.net/1773/6344.
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Popov, Nikita. "Expression and activity of Myc network proteins during cell cycle progression and differentiation /." Sundbyberg, 2004. http://diss.kib.ki.se/2004/91-7349-856-4/.
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Munteanu, Alina. "Computational models to investigate binding mechanisms of regulatory proteins." Doctoral thesis, Humboldt-Universität zu Berlin, 2018. http://dx.doi.org/10.18452/19148.
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Es gibt tausende regulatorische Proteine in Eukaryoten, die spezifische cis-regulatorischen Elemente von Genen und/oder RNA-Transkripten binden und die Genexpession koordinieren. Auf DNA-Ebene modulieren Transkriptionsfaktoren (TFs) die Initiation der Transkription, während auf RNA-Ebene RNA-bindende Proteine (RBPs) viele Aspekte des RNA-Metabolismus und der RNA-Funktion regulieren. Für hunderte dieser regulatorischer Proteine wurden die gebundenen Gene beziehungsweise RNA-Transkripte, sowie deren etwaige Sequenzbindepräferenzen mittels in vivo oder in vitro Hochdurchsatz-Experimente bestimmt. Zu diesen Methoden zählen unter anderem Chromatin-Immunpräzipitation (ChIP) gefolgt von Sequenzierung (ChIP-seq) und Protein Binding Microarrays (PBMs) für TFs, sowie Cross-Linking und Immunpräzipitation (CLIP)-Techniken und RNAcompete für RBPs. In vielen Fällen kann die zum Teil hohe Bindespezifität für ein zumeist sehr kurzes Sequenzmotiv regulatorischer Proteine nicht allein durch die gebundene Primärsequenz erklärt werden.
Um besser zu verstehen, wie verschiedene Proteine ihre regulatorische Spezifität erreichen, haben wir zwei Computerprogramme entwickelt, die zusätzliche Informationen in die Analyse von experimentell bestimmten Bindestellen einbeziehen und somit differenziertere Bindevorhersagen ermöglichen. Für Protein-DNA-Interaktionen untersuchen wir die Bindungsspezifität paraloger TFs (d.h. Mitglieder der gleichen TF-Familie). Mit dem Fokus auf der Unterscheidung von genomischen Regionen, die in vivo von Paaren eng miteinander verwandter TFs gebunden sind, haben wir ein Klassifikationsframework entwickelt, das potenzielle Co-Faktoren identifiziert, die zur Spezifität paraloger TFs beitragen. Für Protein-RNA-Interaktionen untersuchen wir die Rolle von RNA-Sekundärstruktur und ihre Auswirkung auf die Auswahl von Bindestellen. Wir haben einen Motif-Finding-Algorithmus entwickelt, der Sekundärstruktur und Primärsequenz integriert, um Bindungspräferenzen der RBPs besser zu bestimmen.There are thousands of eukaryotic regulatory proteins that bind to specific cis regulatory regions of genes and/or RNA transcripts and coordinate gene expression. At the DNA level, transcription factors (TFs) modulate the initiation of transcription, while at the RNA level, RNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. The DNA or RNA targets and/or the sequence preferences of hundreds of eukaryotic regulatory proteins have been determined thus far using high-throughput in vivo and in vitro experiments, such as chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) and protein binding microarrays (PBMs) for TFs, or cross-linking and immunoprecipitation (CLIP) techniques and RNAcompete for RBPs. However, the derived short sequence motifs do not fully explain the highly specific binding of these regulatory proteins. In order to improve our understanding of how different proteins achieve their regulatory specificity, we developed two computational tools that incorporate additional information in the analysis of experimentally determined binding sites. For protein-DNA interactions, we investigate the binding specificity of paralogous TFs (i.e. members of the same TF family). Focusing on distinguishing between genomic regions bound in vivo by pairs of closely-related TFs, we developed a classification framework that identifies putative co-factors that provide specificity to paralogous TFs. For protein-RNA interactions, we investigate the role of RNA secondary structure and its impact on binding-site recognition. We developed a motif finding algorithm that integrates secondary structure together with primary sequence in order to better identify binding preferences of RBPs.
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Ruhlman, Tracey. "Determinants of Chloroplast Gene Expression and Applications of Chloroplast Transformation in Lactuca Sativa and Nicotiana Tabacum." Doctoral diss., University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2854.
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Genetic modification of plastids in the model plant tobacco (Nicotiana tabacum) has demonstrated that numerous foreign gene products can accumulate to high levels in this setting. Plastid biotechnology is maturing to encompass the improvement of food and feed species and the production of biopharmaceutical proteins for oral delivery necessitating development of stable transplastomic edible plants. In the interest of establishing an edible platform we have investigated the use of native and foreign regulatory elements in relation to foreign gene expression in plastids. Multiple sequence alignments of intergenic regions for 20 species of angiosperm showed that despite 95% identity in the coding region, identity in the psbA upstream region is 59% across all taxa examined, other gene coding regions displayed sequence identity of 80-97%, whereas the non-coding regions were 45-79% suggesting that our physical data can be extrapolated beyond the model presented. We found that by exchanging psbA untranslated regions (UTRs) between N. tabacum and lettuce (Lactuca sativa), the expression of the CTB-proinsulin (CTB-Pins) monocistronic transcript declined by 84% and foreign protein accumulation was reduced by as much as 97% in mature leaves. Polyribosome association assays suggest that ribosome-free transgenic transcripts are stabilized where the native UTR is employed. RNA EMSA revealed that binding proteins interacted with psbA 5' UTRs in a species specific manner and the half life of the L. sativa 5'UTR-CTB-Pins mRNA was reduced by 3.7 fold in N. tabacum stromal extracts. Our data indicate that the use of species-specific regulatory elements could lead to establishment of reproducible plastid transformation in desirable target species such as L. sativa. Using transplastomic L. sativa for oral delivery of bioencapsulated CTB-Pins we delayed the onset of diabetes in NOD mice when retinyl acetate supplement was provided compared to untouched mice. In this 30 week study we monitored blood glucose levels and evaluated the in vitro suppressive capacity of regulatory T cells isolated from diabetic mice. Whether delay or prevention was achieved appeared to be a function of antigen dose as high dose resulted in a nine week delay of onset while low dose reduced the incidence of diabetes by 36%. In addition we have evaluated metabolic engineering in the N. tabacum model where we generated cis-genic lines expressing nucleus-encoded methionine pathway enzymes in plastids. Transplastomic expression of Cystathionine gamma-Synthase led to a three-fold increase in enzyme activity and a doubling of methionine content in leaves without a deleterious phenotype. In exploring molecular mechanisms supporting gene expression in plastids and applying transplastomic technology to real human problems this work seeks address the potential of plastid biotechnology for improvement of commodity crops and production of biopharmaceuticals.Ph.D.
Department of Biomolecular Science
Other
Biomedical Sciences PhD
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Nordzell, Mariette. "Functional studies on the nuclear receptor Nurr1 /." Stockholm : Karolinska institutet, 2004. http://diss.kib.ki.se/2004/91-7349-867-X/.
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Richter, Jennifer Jo. "Gene expression during skeletal muscle development affect of in ovo IGF-1 administration on broiler embryogenesis and postnatal myogenesis in the mouse /." Morgantown, W. Va. : [West Virginia University Libraries], 2002. http://etd.wvu.edu/templates/showETD.cfm?recnum=2517.
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Thesis (Ph. D.)--West Virginia University, 2002.Title from document title page. Document formatted into pages; contains xii, 118 p. : ill. (some col.). Includes abstract. Includes bibliographical references.
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Hallal, Samantha. "Characterisation of the zinc fingers of erythroid krüppel-like factor." Connect to full text, 2008. http://ses.library.usyd.edu.au/handle/2123/4030.
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Thesis (Ph. D.)--University of Sydney, 2009.Title from title screen (viewed February 10, 2009). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular and Microbial Biosciences, Faculty of Science. Degree awarded 2009; thesis submitted 2008. Includes bibliographical references. Also available in print form.
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Fagerström, Billai Fredrik. "Genome wide analysis of the Ssn6-Tup11/Tup12 co-repressor complex in the fission yeast Schizosaccharomyces pombe /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-120-3/.
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Spinler, Jennifer K. "Characterizing the diphtheria-toxin-repressor (DtxR) regulon in Corynebacterium diphtheriae /." Connect to full text via ProQuest. IP filtered, 2006.
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Thesis (Ph.D. in Microbiology) -- University of Colorado at Denver and Health Sciences Center, 2006.Typescript. Includes bibliographical references (leaves 142-160). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Carroll, Michelle. "Inhibitor of DNA binding (ID) proteins in the epididymis and the impact of Id deficiency on gene expression, spermatozoal motility, and progeny outcome." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=106287.
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The epididymis is a single, highly convoluted tubule linking the efferent ducts of the testis to the vas deferens. It is the site where sperm mature, acquiring forward motility and fertilizing ability. Spermatozoal maturation is the result of complex, dynamic interactions between spermatozoa and the highly specialized, continuously changing luminal microenvironment established by the epididymal epithelium. Regionalized gene expression is paramount to the establishment of this microenvironment. The goal of this thesis was to examine the ID family of transcriptional regulators in the epididymis and their role in male-mediated reproduction. The first objective was to establish the longitudinal expression profiles and cellular distribution of the four IDs along the epididymis and how expression was affected by orchidectomy and by aging. IDs display unique longitudinal expression profiles along the tissue, having highly regionalized expression, and are primarily expressed in non-overlapping cell types. While aging resulted in no noticeable effect, we observed that the expression of ID3 is differentially regulated in response to orchidectomy along the tissue. The next objective was to determine the functional consequences of Id3 deficiency (Id3tm1Cmu/Id3tm1Cmu) on epididymal histology and gene expression, as well as spermatozoal counts and motility parameters. Although Id3 deficiency had no noticeable impact on epididymal histology, the targeted mutation altered several sperm motility parameters. We undertook genome-wide expression profiling to elucidate region-specific effects and found that the predominant effect of the Id3 null mutation is in the cauda epididymidis, where the expression of many transcription factors was significantly affected. The final objective was to assess the fertility status and progeny outcome of animals deficient in Id3. Matings with Id3-/- males resulted in abnormal reproductive outcomes and developmental phenotypes in gestational day 18 fetuses. These were characterized by an elevated rate of fetal death, reduced body weights of live fetuses, a higher rate of external malformations, and aberrant skeletal development. Collectively, these studies indicate that Id3 may contribute to the molecular basis of regionalized gene expression in the epididymis, provide novel insights into the role of Id3 in male-mediated reproduction, and afford new perspectives for future avenues of research.L'épididyme est un tubule unique, replié maintes fois sur lui-même, reliant les canaux efférents des testicules au canal déférent. L'épididyme est le site de maturation des spermatozoïdes; là où ils acquièrent leur mobilité et leur habileté à fertiliser. Cette maturation est le résultat d'une dynamique complexe d'interactions entre les spermatozoïdes et un microenvironnement hautement spécialisé, continuellement entretenu par l'épithélium de l'épididyme. L'expression régionalisée des gènes de cet organe est primordiale pour le maintien de ce microenvironnement. Le but de ces travaux de thèse était d'étudier la famille des régulateurs de transcription que sont les inhibiteurs de liaison à l'ADN (IDs), dans l'épididyme ainsi que leur rôle en reproduction mâle. Le premier objectif, était d'établir le profil d'expression longitudinal et cellulaire des IDs dans l'épididyme et d'observer comment l'orchidectomie et l'âge pouvaient affecter ces profils d'expression et de localisation. Les protéines IDs montrent une expression longitudinale unique le long du tissu, un profil régionalisé précis, et sont exprimées dans des cellules différentes. Alors que l'âge ne montre aucun effet notable, nous avons observé que l'expression de la protéine ID3 est modifiée après orchidectomie dans l'ensemble de l'épididyme. Le second objectif était de déterminer les conséquences fonctionnelles d'une déficience en ID3 (Id3tm1Cmu/Id3tm1Cmu) sur l'histologie et sur les profils d'expression génétique de l'épididyme, ainsi que les paramètres de numération et de mobilité des spermatozoïdes. Bien que la déficience en ID3 n'ait aucun impact histologique, la mutation affecte les paramètres de mobilité des spermatozoïdes. Pour élucider l'effet de la mutation sur le profil régionalisé d'expression des gènes, nous avons entrepris une analyse du profil d'expression génomique et avons mis en évidence que l'effet de la mutation prédomine dans la partie caudale de l'épididyme, là où l'expression de plusieurs facteurs de transcription était affectée. Enfin, le dernier objectif était d'évaluer la fertilité et la descendance des animaux déficients en ID3. La descendance des souris mâles Id3-/- et leurs phénotypes développementaux sont anormaux. Ces derniers étant caractérisés par un taux élevé de mort fœtale, un poids de naissance faible, un taux de malformations externes anormalement haut, et un développement du squelette anormal. L'ensemble de ces études indique que ID3 contribuerait à la base moléculaire de l'expression régionalisée des gènes de l'épididyme, fournit de nouvelles données sur le rôle de ID3 en reproduction chez le mâle, et offre de nouvelles perspectives pour de futures pistes de recherche.
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Fajardo, Mark A. "Defining cis- and trans-acting components for Prm-1 temporal translational control during murine spermatogenesis /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/10251.
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Clingman, Carina C. "A Feedback Loop Couples Musashi-1 Activity to Omega-9 Fatty Acid Biosynthesis: A Dissertation." eScholarship@UMMS, 2014. http://escholarship.umassmed.edu/gsbs_diss/718.
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All living creatures change their gene expression program in response to nutrient availability and metabolic demands. Nutrients and metabolites can directly control transcription and activate second-‐messenger systems. In bacteria, metabolites also affect post-‐transcriptional regulatory mechanisms, but there are only a few isolated examples of this regulation in eukaryotes. Here, I present evidence that RNA-‐binding by the stem cell translation regulator Musashi-‐1 (MSI1) is allosterically inhibited by 18-‐22 carbon ω-‐9 monounsaturated fatty acids. The fatty acid binds to the N-‐terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. I identify stearoyl-‐CoA desaturase-‐1 as a MSI1 target, revealing a feedback loop between ω-‐9 fatty acid biosynthesis and MSI1 activity. To my knowledge, this is the first example of an RNA-‐binding protein directly regulated by fatty acid. This finding may represent one of the first examples of a potentially broad network connecting metabolism with post-‐transcriptional regulation.
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Pande, Sandhya. "Regulation of Runx Proteins in Human Cancers: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/559.
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Runt related transcription factors (Runx) play an important role in mammalian development by regulating the expression of key genes involved in cell proliferation, differentiation and growth. The work described in this thesis details the mechanisms by which the activity of two members of this family are regulated in human cells. Chapter One provides a brief introduction of Runx transcription factors.
Chapter Two describes the regulation of Runx2 protein by the PI3 kinase/Akt pathway in human breast cancer cells. The PI3 kinase/Akt pathway is one of the major signal transduction pathways through which growth factors influence cell proliferation and survival. It is also one of the most frequently dysregulated pathways in human cancers. We identify Runx2 protein, a key regulator of breast cancer invasion as a novel substrate of Akt kinase and map residues of Runx2 that are phosphorylated by Akt in breast cancer cells. Our results show that phosphorylation by Akt increases the binding of Runx2 protein to its target gene promoters and we identify the phosphorylation events that enhance DNA binding of Runx2. Our work establishes Runx2 protein as a critical effecter downstream of Akt that regulates breast cancer invasion.
In Chapter Three we describe the subnuclear localization of the tumor suppressor protein Runx3 during interphase and mitosis. We find that similar to other Runx family members, Runx3 protein resides in nuclear matrix associated foci during interphase. We delineate a subnuclear targeting signal that directs Runx3 to these nuclear matrix associated foci. Our work establishes that this association of Runx3 protein with the nuclear matrix plays a vital role in regulating its transcriptional activity.
Chromatin immunoprecipitation results show that Runx3 occupies rRNA promoters during interphase. We also find that Runx3 remains associated with chromosomes during mitosis and localizes with nucleolar organizing regions (NORs), reflecting an interaction with epigenetic potential.
This thesis provides novel insights into various mechanisms by which cells regulate the activity of Runx proteins.
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Sundqvist, Anders. "Characterisation of CtBP : A Co-Repressor of Transcription that Interacts with the Adenovirus E1A Protein." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-5144-6/.
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Lublin, Alex Louis. "The pumilio proteins PUF-5 and PUF-6/7/10 are necessary for repression of C. Elegans notch/glp-1 during late oogenesis (or not all that glitters is GLD-1) /." Connect to full text at ProQuest Digital Dissertations. IP filtered, 2005.
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Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005.Typescript. Includes bibliographical references (leaves 82-86). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Åberg, Anna. "New insights into the role of ppGpp and DksA through their effect on transcriptional regulation of housekeeping and colonization related genes of Escherichia coli /." Umeå : Univ, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1669.
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Rupon, Jeremy William. "The Role of DNA Methylation and Methyl Binding Domain Protein 2 in the Regulation of Human Embryonic and Fetal Beta Type Globin Genes." VCU Scholars Compass, 2006. http://hdl.handle.net/10156/1833.
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Tucker, Sean Newton. "Ikaros affects the expression of the interleukin-2 receptor beta chain and lymphoid cell potential /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/8338.
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Gerbracht, Jennifer Victoria [Verfasser], Niels [Gutachter] Gehring, Kay [Gutachter] Hofmann, and Elmar [Gutachter] Wahle. "Analysis of post-transcriptional gene expression modulated by mRNA stability and RNA-binding proteins in human cells / Jennifer Victoria Gerbracht ; Gutachter: Niels Gehring, Kay Hofmann, Elmar Wahle." Köln : Universitäts- und Stadtbibliothek Köln, 2020. http://d-nb.info/1208830554/34.
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Gerbracht, Jennifer V. [Verfasser], Niels [Gutachter] Gehring, Kay [Gutachter] Hofmann, and Elmar [Gutachter] Wahle. "Analysis of post-transcriptional gene expression modulated by mRNA stability and RNA-binding proteins in human cells / Jennifer Victoria Gerbracht ; Gutachter: Niels Gehring, Kay Hofmann, Elmar Wahle." Köln : Universitäts- und Stadtbibliothek Köln, 2020. http://d-nb.info/1208830554/34.
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Holter, Elin. "Modulation of nuclear receptor activity by a unique class of corepressors /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-039-7/.
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Barwe, Sonali P. "Studies On The Molecular Mechanism Of Cytokinin Action: Involvement Of Ca2+, Protein Kinase And Concurrent Protein Synthesis In Signaling Of Cytokinin-Induced Expression Of Pathogenesis-Related Enzymes In Cucumber." Thesis, Indian Institute of Science, 2000. http://hdl.handle.net/2005/178.
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Phytohormones act as signals to regulate plant growth and development by modulation of gene expression in response to internal developmental cues or external environmental stimuli, such as light and pathogen infection. There are five major classes of phytohormones, viz. auxins, cytokinins, gibberellins, ethylene and abscisic acid. Of these, cytokinins, 6N substituted adenine derivatives, are of special importance owing to their possible diverse roles in plant growth and development. They induce cell division, cell expansion in cotyledon, chloroplast and etioplast development, suppression of apical dominance and senescence, and differentiation of in vitro cultured cells. However, very little is known about the mechanism of cytokinin action at the molecular level. Cytokinins have been demonstrated to modulate the expression of genes coding for several enzymes including nitrate reductase, ribulose-l,5-bisphosphate carboxylase, RNA polymerase I, and pathogenesis-related (PR) enzymes, i.e. chitinases and β-1,3- glucanases. One of the important questions regarding cytokinin regulation of enzyme activities and/or the accumulation of their corresponding proteins and mRNAs is how the cytokinin signal is transduced.
There is considerable evidence from earlier reports demonstrating that pathogens alter hormone physiology of the host plant and it has been proposed that the infection-associated enzyme changes might be mediated by phytohormones. In the present study, two PR enzymes, viz. cucumber chitinase and β-l,3-glucanase, have been chosen to examine the mode of regulation of their gene expression by cytokinins, including the identification of cytokinin signal transduction components. Plant chitinases and glucanases are important enzymes in plant defense mechanisms against fungal pathogens as they degrade the major fungal cell wall components, chitin and β-1,3-glucan, respectively. Besides their role in plant defense, they are known to be involved in diverse physiological and developmental processes, such as embryogenesis, seed germination and flower development, and are also developmentally and hormonally regulated.
Initially, in order to study the effects of various cytokinins on chitinase and β-1,3-gIucanase enzyme activities and their gene expression, cotyledons excised from seven-day-old dark-grown cucumber seedlings were treated with water, and cytokinins, viz. benzyladenine, kinetin, zeatin and zeatin riboside. It was observed that chitinase and β-l,3-ghucanase enzyme activities and their transcripts were induced to varying extents following treatments of cotyledons with the cytokinins tested. However, a maximum increase in enzyme activities and their transcript levels was noticed in zeatin-treated cotyledons. Therefore, zeatin was used for further studies.
The main objective of the present study was to investigate the cytokinin signal transduction mechanism involving the induction of expression of chitinase and β-1-3-glucanase. In order to obtain insights into the downstream components of the cytokinin-signaling pathway, effects of several agonists and antagonists of the signal transduction components on zeatin-induced chitinase and β-l,3-glucanase activities, and their protein and transcript levels were monitored by enzyme assay, by immunoblot analysis, and by northern analysis, respectively. Treatment of excised dark-grown cucumber cotyledons with staurosporine, a broad spectrum protein kinase inhibitor, reduced the zeatin-induced chitinase and β-l,3-glucanase enzyme activities and the accumulation of their proteins and transcripts. On the other hand, treatment with sodium fluoride, a general inhibitor of protein phosphatases, stimulated the basal chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation, whereas it had no effect on the zeatin-induced enzyme activities and their protein and transcript accumulation. These findings suggested that protein phosphorylation is critical in the cytokinin induction of expression of chitinase and β-l,3-glucanase.
Since Ca2+ is known to be an important second messenger in several plant signal transduction pathways, the possible involvement of Ca2+ in the cytokinin-induced expression of chitinase andβ-l,3-glucanase was examined. The results of the present investigation showed that the chitinase and β-1,3-ghicanase activities and their proteins and transcripts were appreciably increased by exogenous CaCl2 treatment in control cotyledons. Treatment of cotyledons with zeatin plus CaCl2 did not result in a further increase in either these enzyme activities or their protein and transcript accumulation as compared to zeatin or CaCl2 treatment alone. The lack of additivity of zeatin plus CaCl2 treatment indicated a common mechanism of action of zeatin and Ca2+ in the induction of these enzyme activities and their gene expression. To test the occurrence of influx of extracellular Ca2+ by cytokinin, cotyledons were treated with the plasma membrane Ca2+ channel blocker, verapamil, and Ca2+ ionophore A23187. Verapamil treatment inhibited the zeatin-induced chitinase and β-1,3-ghicanase enzyme activities and their protein and transcript accumulation. An increase in the intracellular Ca2+ levels by means of Ca2+ ionophore treatment resulted in a significant increase in basal chitinase and β-l,3-glucanase activities and their protein and transcript accumulation. These results suggested that an influx of extracellular Ca2+ leading to increased levels of cytosolic Ca2+ is required for the cytokinin induction of expression of these enzymes.
The correlation of chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation in the zeatin-treated cotyledons suggested that the cytokinin zeatin stimulates chitinase and β-l,3-glucanase accumulation at the mRNA level and that the increase in enzyme activities is due to an increase in the amount of the enzyme protein and not by the activation of the existing enzyme. Further, the effect of zeatin on both the enzyme activities and their transcript levels under conditions that inhibit protein synthesis was studied. Treatment of excised dark-grown cucumber cotyledons with cycloheximide, an inhibitor of protein synthesis, in the presence of zeatin, completely nullified the stimulatory effect of zeatin. These results indicated the requirement of cytokinin-induced enhanced concurrent protein synthesis in the observed stimulation of chitinase and β-l,3-glucanase enzyme activities as well as their transcript accumulation Ca2+
In an attempt to isolate the full length cucumber β-l,3-glucanase cDNA from a cucumber cDNA library, we isolated and sequenced one cDNA clone, which was 978 bp long and had a potential polyadenylation signal A ATA A starting 172 bases before the polyadenylation tail A deduced amino acid sequence of the cDNA, which was 242
amino acids in length, apparently encoded a partial β-amyrin synthase. Sequence comparison of the deduced partial amino acid sequence of cucumber β-amyrin synthase with other known plant β-amyrin synthase sequences available in databases revealed significant homologies to β-amyrin synthases from Panax, Pisum and Glycyrrhiza. Southern blot analysis indicated that there was only one β-amyrin synthase gene in the cucumber genome. RT-PCR analysis performed on total RNA isolated from zeatin- and salicylic acid-treated cotyledons using forward and reverse primers designed from the internal regions of the cDNA showed that the transcript levels of β-amyrin synthase were enhanced by both zeatin and salicylic acid.
In conclusion, we have demonstrated that chitinase and β-l,3-glucanase accumulation is stimulated by exogenous cytokinin treatment of excised cucumber cotyledons, and this effect is correlated with the content of chitinase and β-1,3-glucanase transcripts as judged by northern analyses. Further, the findings reported in the thesis suggested that Ca2+ influx from extracellular space, protein phosphorylation by staurosporine-sensitive protein kinase(s) and concurrent protein synthesis are required for the signaling of cytokinin-induced expression of both these pathogenesis-related enzymes.
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Almqvist, Jenny. "Epstein-Barr virus nuclear antigen 1, Oct & Groucho/TLE in control of promoter regulation /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-523-2/.
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Nielsen, Mark David. "Regulation of two subfamilies of adenylyl cyclase by Gi-coupled receptors : a possible role during cAMP-dependent synaptic plasticity in the Hippocampus /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/6247.
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Young, Daniel W. "Regulation of Cell Growth and Differentiation within the Context of Nuclear Architecture by the Runx2 Transcription Factor: a Dissertation." eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/19.
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The Runx family of transcription factors performs an essential role in animal development by controlling gene expression programs that mediate cell proliferation, growth and differentiation. The work described in this thesis is concerned with understanding mechanisms by which Runx proteins support this program of gene expression within the architectural context of the mammalian cell nucleus. Multiple aspects of nuclear architecture are influenced by Runx2 proteins including sequence-specific DNA binding at gene regulatory regions, organization of promoter chromatin structure, and higher-order compartmentalization of proteins in nuclear foci. This work provides evidence for several functional activities of Runx2 in relation to architectural parameters of gene. expression for the control of cell growth and differentiation. First, the coordination of SWI/SNF mediated chromatin alterations by Runx2 proteins is found to be a critical component of osteoblast differentiation for skeletal development. Several chromatin modifying enzymes and signaling factors interact with the developmentally essential Runx2 C-terminus. A patent-pending microscopic image analysis strategy invented as part of this thesis work - called intranuclear informatics - has contributed to defining the C-terminal portion of Runx2 as a molecular determinant for the nuclear organization of Runx2 foci and directly links Runx2 function with its organization in the nucleus. Intranuclear informatics also led to the discovery that nuclear organization of Runx2 foci is equivalently restored in progeny cells following mitotic division - a natural perturbation in nuclear structure and function. Additional microscopic studies revealed the sequential and selective reorganization of transcriptional regulators and RNA processing factors during progression of cell division to render progeny cells equivalently competent to support Runx2 mediated gene expression. Molecular studies provide evidence that the Runx proteins have an active role in retaining phenotype by interacting with target gene promoters through sequence-specific DNA binding during cell division to support lineage-specific control of transcriptional programs in progeny cells. Immunolocalization of Runx2 foci on mitotic chromosome spreads revealed several large foci with pairwise symmetry on sister chromatids; these foci co-localize with the RNA polymerase I transcription factor, Upstream Binding Factor (UBFl) at nucleolar organizing regions. A series of experiments were carried out to reveal that Runx2 interacts directly with ribosomal DNA loci in a cell cycle dependent manner; that Runx2 is localized to UBF foci within nucleoli during interphase; that Runx2 attenuates rRNA synthesis; and that this repression of ribosomal gene expression by Runx2 is associated with cell growth inhibition and induction of osteoblast-specific gene expression. This thesis has identified multiple novel mechanisms by which Runx2 proteins function within the hierarchy of nuclear architecture to control cell proliferation, growth and differentiation.
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Simpson, Raina Jui Yu. "The multiple roles of zinc finger domains." University of Sydney. Molecular and Microbial Biosciences, 2004. http://hdl.handle.net/2123/655.
Full textAbstract:
Zinc finger (ZnF) domains are prevalent in eukaryotes and play crucial roles in mediating protein-DNA and protein-protein interactions. This Thesis focuses on the molecular details underlying interactions mediated by two ZnF domains. The GATA-1 protein is vital for the development of erythrocytes and megakaryocytes. Pertinent to the protein function is the N-terminal ZnF. In particular, this domain mediates interaction with DNA containing GATC motifs and the coactivator protein FOG. The importance of these interactions was illustrated by the findings in Chapter 3 that naturally occurring mutations identified in patients suffering from blood disorders affect the interaction of the N-terminal ZnF with either DNA (R216Q mutation) or FOG (V205M and G208S mutations). In addition to the interaction FOG makes with GATA-1, it also interacts with the centrosomal protein TACC3. In Chapter 4, this interaction is characterised in detail. The solution structure of the region of FOG responsible for the interaction is determined using NMR spectroscopy, revealing that it is a true classical zinc finger, and characterisation of the interaction domain of TACC3 showed that the region is a dimeric coiled-coil. The FOG:TACC3 interaction appears to be mediated by a-helices from the two proteins. The data presented here represent some of the first described molecular details of how a classical ZnF can contact a protein partner. Interestingly, the a-helix used by the FOG finger to bind TACC3 is the same region utilised by DNA-binding classical zinc fingers to contact DNA. In addition to the multiple roles played by ZnFs, this domain is also known for its robustness and versatility. In Chapter 5, incomplete ZnF sequences were assessed for its ability to form functional zinc-binding domains. Remarkably, CCHX sequences (in the context of BKLF finger 3) were able to form discrete zinc-binding domains and also, mediate both protein-DNA and protein-protein interactions. This result not only illustrates the robust nature of ZnFs, it highlights the need for expanding ZnF sequence criteria when searching for functional zinc-binding modules. Together, the data presented here help further our understanding of zinc finger domains. Similar to the use of DNA-binding ZnFs in designer proteins, these data may start us on the path of designing novel protein-binding ZnFs.