Myelin Basic Protein is a major structural protein of vertebrate myelin. The gene that codes for MBP is contained within the golli-MBP complex. This gene complex consists of two overlapping transcription units, golli and MBP, which are regulated by two distinct promoters. The golli unit is expressed in cells of the oligodendrocyte lineage (Central Nervous System), neurons, B and T lymphocytes, testis and thymus. However, the MBP unit is expressed exclusively in oligodendrocytes and Schwann cells (Peripheral Nervous System). The expression of the MBP unit is regulated mainly at the level of transcription by proteins that bind in a specific manner to DNA sequences located within its promoter region. The identification of these proteins and DNA sequences is essential to understanding the mechanisms that regulate the transcription of the MBP unit. This project was initiated by the isolation of the putative promoter region of the mouse myelin basic protein (MBP) gene. To achieve this the Hind III - Sac I fragment of pEX1 plasmid was subcloned in the vector pBluescript. The cloned insert, which corresponds to the region between nucleotides -1319 and +227 relative to the transcription start site of the mouse MBP gene, was subsequently sequenced manually using the chain termination method. Sequence analysis revealed a number of putative binding sites for transcription factors. The region -609 to -577 was selected for further studies because work published by other groups suggested that it contains a cell-type specific (for oligodendrocytes) transcription activator. The presence of protein factors specifically binding to the region -609 to -577 was demonstrated by electrophoretic mobility shift assay (EMSA).For this purpose, nuclear extracts were prepared from rodent brain or established glial cell lines e.g. C6 glioma cells. Extracts from tissues and cell lines, which do not express myelin basic protein e.g., HeLa cells served as a control. Nuclei were isolated by Dounce homogenisation of cultured cells or brain tissue. The proteins were then isolated by high salt extraction of the nuclei followed by ammonium sulphate fractionation. Putative protein(s) binding to the region located between nucleotides -609 to -577 of the myelin basic protein gene promoter were identified using the yeast one-hybrid system. This assay is based on the interaction between a specific protein DNA binding domain and the target DNA sequence. Proteins are expressed as fusions to the GAL4 activation domain (AD) in the yeast reporter strain in which the target sequence has been inserted upstream of the HIS3 gene minimal promoter. Binding of AD fusions to the target sequence increases activity of the HIS3 promoter enabling growth on medium lacking histidine. In this work a yeast reporter strain containing four copies of the -609 to -577 region tandemly repeated upstream of the HIS3 gene minimal promoter was constructed. A library containing rat brain cDNAs fused to the activation domain of GAL4 was screened using this strain as a host. Seven clones were obtained on medium lacking histidine in the presence of 30 mM 3-aminotriazole. DNA from these clones was automatically sequenced and analysed for sequence homology with known transcription factors by comparing the nucleotide and protein sequences to EMBL/Genbank and Swissprot/Swall databases using theFastA and Blast search tools. From the results of the homology searches the clones were identified as follows: the activating transcription factor 2 (ATF-2), the pituitary specific positive transcription factor 1 (Pit-1) or general transcription factor 2i, the E2F family transcription factor, the PASK protein and two of the clones were identified as c-jun. One clone, however, remains unidentified and this could be a novel transcription factor.

As quinases dependentes de ciclinas são proteínas que podem ser divididas de acordo com a sua atuação no ciclo celular ou no controle transcricional, elas se tornam ativas em determinadas etapas do ciclo celular dependendo do seu grau de fosforilação e de sua ligação com ciclinas e proteínas inibitórias, e exercem sua função fosforilando outras proteínas envolvidas no ciclo de divisão celular e transcrição influenciando suas atividades, garantindo que cada processo do ciclo ocorra em uma sequência ordenada. A CDK13 faz parte da família de proteínas quinases dependentes de ciclina, pode se ligar a ciclinas do tipo L ou K, regula os eventos de \"splicing\" alternativo, e interage com a proteína Tat do vírus HIV atuando como um possível fator de restrição, sendo que sua superexpressão diminui a produção de algumas proteínas virais suprimindo a produção do vírus. O DNA referente à CDK13 é replicado em células cancerosas, principalmente dos tipos hepático e cólon e reto, sendo um alvo para inibidores para tratamento de câncer. A fim de contribuir para o estudo dessa proteína, o projeto tem como objetivo expressá-la utilizando métodos de tecnologia de DNA recombinante. A sequência de DNA referente à CDK13 foi amplificada pela reação em cadeia da polimerase, após sua purificação, foi inserida no vetor pCR-Blunt e clonada em células de E. coli DH5α competentes. Porém, o DNA não foi liberado pela reação com as enzimas de restrição BamHI e NdeI. As bactérias Rosetta(DE3) transformadas com um plasmídeo sintético e crescidas em meio de auto-indução expressaram a CDK13. Após lise celular e purificação em coluna de Ni2+, a proteína foi detectada por Western Blot. Já as bactérias Rosetta(DE3) transformadas com o plasmídeo sintético modificado (o qual compreende a região do DNA que expressa o bolsão de ligação da CDK13), e induzidas em meio LB expressaram a CDK13, porém não foi possível purificá-la em coluna de afinidade ao Ni2+. / The cyclin-dependent kinases are proteins that can be classified by their function in the cell cycle or transcriptional control. They are activated in particular steps of the cell cycle depending on their phosphorylation degree, cyclin binding and inhibitory proteins. They act phosphorylating other proteins involved in the cell cycle and transcriptional control, influencing in their activities, ensuring that each step of the cell cycle occur in an ordered sequence. The CDK13 is one of the cyclin-dependent kinases family member, it can bind to L or K cyclins, regulates the alternative splicing and interact with HIV Tat protein, acting as a possible restriction factor, its overexpression decreases the production of some viral proteins, and suppresses the virus production. The DNA corresponding to CDK13 is replicated in cancer cells, mainly of hepatic and colon rectal types; therefore it is a target for inhibitors for cancer therapy. In order to contribute for the studies of this protein, the goal of the project is to express it using methods of recombinant DNA technology. The DNA sequence corresponding to CDK13 was amplified by polymerase chain reaction, after its purification, it was inserted to pCR-Blunt vector and cloned into E. coli DH5α competent cells. However, the DNA wasn\'t released by the BamHI and NdeI restriction enzymes. The Rosetta(DE3) cells transformed with a synthetic plasmid pET28a::CDK13 and grown in auto-induction media expressed the CDK13. After cell lysis and purification by Ni2+ affinity colum, the protein was identified by Western Blot. However, the Rosetta(DE3) cells transformed with the modified synthetic plasmid (that comprehends the DNA region which expresses the binding pocket region) induced in LB media, expressed the CDK13. Yet, it wasn\'t possible to purify the protein in the Ni2+ affinity column.

As quinases dependentes de ciclinas são proteínas que podem ser divididas de acordo com a sua atuação no ciclo celular ou no controle transcricional, elas se tornam ativas em determinadas etapas do ciclo celular dependendo do seu grau de fosforilação e de sua ligação com ciclinas e proteínas inibitórias, e exercem sua função fosforilando outras proteínas envolvidas no ciclo de divisão celular e transcrição influenciando suas atividades, garantindo que cada processo do ciclo ocorra em uma sequência ordenada. A CDK13 faz parte da família de proteínas quinases dependentes de ciclina, pode se ligar a ciclinas do tipo L ou K, regula os eventos de \"splicing\" alternativo, e interage com a proteína Tat do vírus HIV atuando como um possível fator de restrição, sendo que sua superexpressão diminui a produção de algumas proteínas virais suprimindo a produção do vírus. O DNA referente à CDK13 é replicado em células cancerosas, principalmente dos tipos hepático e cólon e reto, sendo um alvo para inibidores para tratamento de câncer. A fim de contribuir para o estudo dessa proteína, o projeto tem como objetivo expressá-la utilizando métodos de tecnologia de DNA recombinante. A sequência de DNA referente à CDK13 foi amplificada pela reação em cadeia da polimerase, após sua purificação, foi inserida no vetor pCR-Blunt e clonada em células de E. coli DH5α competentes. Porém, o DNA não foi liberado pela reação com as enzimas de restrição BamHI e NdeI. As bactérias Rosetta(DE3) transformadas com um plasmídeo sintético e crescidas em meio de auto-indução expressaram a CDK13. Após lise celular e purificação em coluna de Ni2+, a proteína foi detectada por Western Blot. Já as bactérias Rosetta(DE3) transformadas com o plasmídeo sintético modificado (o qual compreende a região do DNA que expressa o bolsão de ligação da CDK13), e induzidas em meio LB expressaram a CDK13, porém não foi possível purificá-la em coluna de afinidade ao Ni2+. / The cyclin-dependent kinases are proteins that can be classified by their function in the cell cycle or transcriptional control. They are activated in particular steps of the cell cycle depending on their phosphorylation degree, cyclin binding and inhibitory proteins. They act phosphorylating other proteins involved in the cell cycle and transcriptional control, influencing in their activities, ensuring that each step of the cell cycle occur in an ordered sequence. The CDK13 is one of the cyclin-dependent kinases family member, it can bind to L or K cyclins, regulates the alternative splicing and interact with HIV Tat protein, acting as a possible restriction factor, its overexpression decreases the production of some viral proteins, and suppresses the virus production. The DNA corresponding to CDK13 is replicated in cancer cells, mainly of hepatic and colon rectal types; therefore it is a target for inhibitors for cancer therapy. In order to contribute for the studies of this protein, the goal of the project is to express it using methods of recombinant DNA technology. The DNA sequence corresponding to CDK13 was amplified by polymerase chain reaction, after its purification, it was inserted to pCR-Blunt vector and cloned into E. coli DH5α competent cells. However, the DNA wasn\'t released by the BamHI and NdeI restriction enzymes. The Rosetta(DE3) cells transformed with a synthetic plasmid pET28a::CDK13 and grown in auto-induction media expressed the CDK13. After cell lysis and purification by Ni2+ affinity colum, the protein was identified by Western Blot. However, the Rosetta(DE3) cells transformed with the modified synthetic plasmid (that comprehends the DNA region which expresses the binding pocket region) induced in LB media, expressed the CDK13. Yet, it wasn\'t possible to purify the protein in the Ni2+ affinity column.

Ph.D. / Biochemistry and Molecular Biology / The Bacillus subtilis spx gene encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It resides in the yjbC-spx operon and is transcribed from at least four promoters, three (P[subscript]1, P[subscript]2 and P[subscript]B) residing upstream of yjbC and one (P[subscript]M) located in the intergenic region between yjbC and spx. We uncovered a second intergenic promoter, P[subscript]3, from which transcription is elevated in cells treated with the thiol-specific oxidant diamide, by primer extension analysis. P[subscript]3 is recognized by the σ[superscript]A form of RNA polymerase (RNAP) in vitro without the involvement of a transcriptional activator. Deletion analysis together with point mutation analysis uncovered two negative cis-acting control elements within the P[subscript]3 promoter. Previously published studies and transcription factor/transformation array technology uncovered two transcriptional repressors, PerR and YodB that were potential candidates for the missing trans-acting factors affecting P[subscript]3 promoter utilization. PerR was previously characterized as the regulator of the inducible peroxide stress response in B. subtilis, while YodB is a novel DUF24/MarR type repressor that controls genes that are induced in response to phenolic compounds and oxidative stress. The derepression of spx was detected in both perR and yodB mutants by examining the level of spx expression using the spx-bgaB fusion construct. The additive effect was observed in the perR yodB double mutant. The regions of spx P[subscript]3 DNA required for transcriptional repression by YodB and PerR were confirmed by DNase I footprinting analysis. PerR protects an area from approximately position -3 to +35. YodB binds a region from approximately positions -3 to -32. The binding of YodB and PerR proteins to spx P[subscript]3 promoter DNA was impaired by addition of diamide and H[subscript]2O[subscript]2 in vitro as determined by DNase I footprinting analysis. Besides spx, YodB also controls the divergently transcribed yodC gene which encodes a putative nitroreductase that is induced by disulfide stress. Microarray and proteome analyses were performed to identify other genes controlled by YodB. yocJ (azoR1), encoding the putative FMN-dependent NADH-azoreductase, was the most strongly derepressed by yodB null mutation and was induced in response to diamide, catechol, MHQ and nitrofurantoin stress. bsrB encoding a small 6S RNA located downstream of azoR1, is co-transcribed with azoR1 and increased in concentration in response to thiol-reactive compounds. The yodB mutant confers a catechol and MHQ resistance phenotype due to AzoR1 overproduction. In addition, the yodBmhqR double mutant, bearing the deletion of the mhqR gene encoding a MarR-like repressor, that overproduces AzoR1 and MhqR-regulated paralog AzoR2, exhibits hyper-resistance to thiol-reactive compounds. Thus, the detoxification of thiol-reactive substances in YodB and MhqR regulons show overlapping functions. DNase I footprinting analysis, together with promoter sequence alignments, uncovered YodB boxes which contain a common 15 bp consensus sequence for YodB-DNA interaction. The YodB protein contains three cysteine residues Cys6, Cys101 and Cys108. The conserved Cys6 contributes to the repression of spx and azoR1 transcription by YodB. Moreover, mass spectrometry revealed YodB Cys modifications by catechol and MHQ.

AgaR, a protein in <i>Escherichia coli</i> thought to control the metabolism of N-acetylgalactosamine, is a member of the DeoR family of transcriptional regulators. Three transcriptional promoters within a cluster of genes containing the gene for AgaR were identified, specific for <i>agaR, agaZ</i> and <i>agaS</i>, and the transcription start sites mapped. Transcription from these promoters was specifically induced by N-acetylgalactosamine or galactosamine, though K-12 strains lacked the ability to utilize these as sole sources of carbon. The activity of these promoters was constitutively elevated in a strain in which <i>agaR</i> had been disrupted confirming that the promoters are subject to negative regulation by AgaR. AgaR-His6, purified using immobilized metal affinity chromatography, was used for DNase I footprint analysis of the promoter regions. Four operator sites bound by AgaR were identified. A putative consensus binding sequence for AgaR was proposed based on these four sites. <i>In vivo</i> and <i>in vitro</i> analysis of the <i>agaZ</i> promoter indicated that this promoter was activated by the cAMP-cAMP receptor protein (CRP). Expression from the <i>aga</i> promoters was less sensitive to catabolite repression in revertants capable of <i>N</i>-acetylgalactosamine utilization, suggesting that these revertants have mutation(s) that result in an elevated level of inducer for AgaR. A cluster of genes at minute 87.7 of the <i>E. coli</i> genome contains a gene that encodes another member of the DeoR family of transcriptional regulators. This protein, YihW, is more similar to GlpR, transcriptional regulator of <i>sn</i>-glycerol 3-phosphate metabolism in <i>E. coli</i>, than other members of the DeoR family. Despite the high degree of similarity, YihW lacked the ability to repress P_glpK, a promoter known to be controlled by GlpR. A variant of YihW containing substitutions in the putative recognition helix to more closely match the recognition helix of GlpR was also unable to repress P_glpK. Transcriptional promoters identified in this cluster of genes were negatively regulated by YihW. Regulation of genes involved in the metabolism of <i>sn</i>-glycerol 3-phosphate in <i>E. coli</i> by GlpR has been well characterized. However, the function of a protein (GlpE) encoded by a gene cotranscribed with that for GlpR was unknown prior to this work. GlpE was identified as a single-domain, 12-kDa rhodanese (thiosulfate:cyanide sulfurtransferase). The enzyme was purified to near homogeneity and characterized. As shown for other characterized rhodaneses, kinetic analysis revealed that catalysis occurs via an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. K_m (SSO₃²⁻) and K_m (CN⁻) were determined to be 78 mM and 17 mM, respectively. The native molecular mass of GlpE was 22.5 kDa indicating that GlpE functions as a dimer. GlpE exhibited a kcat of 230 s-1. Thioredoxin, a small multifunctional dithiol protein, served as sulfur-acceptor substrate for GlpE with an apparent K_m of 34 mM when thiosulfate was near its K_m, suggesting thioredoxin may be a physiological substrate. / Ph. D.

Protéines difficiles à exprimer: un goulot d'étranglement pour la plupart des biologistes. J'ai choisi d'utiliser comme modèle d’étude Acanthamoeba polyphaga Mimivirus. Ce virus géant à ADN possède des protéines subissant des modifications post-traductionnelles, des structures multi-protéiques ou encore des voies enzymatiques jamais identifiées auparavant dans un virus, ce qui en font un modèle idéal pour l’étude de protéines récalcitrantes. Le but ultime de cette thèse, était de produire les protéines de capsides de Mimivirus. Le rôle de la protéine de capside dans l’assemblage de la particule virale, son infectivité et ses caractéristiques moléculaires sont d’une grande importance. Pour aller du gène à la protéine, J’ai participé à la compréhension de ce qui gouverne la terminaison de la transcription de Mimivirus et également participé à l'analyse globale du transcriptome au cours du cycle d'infection des amibes par Mimivirus. Nous avons montré que les transcrits de Mimivirus sont systématiquement polyadénylés dans des régions formant une structure secondaire en tige-boucle, même s’il n’existe pas de signal de polyadénylation canonique en amont. Nous en avons conclu que la polyadénylation de Mimivirus suit exclusivement une règle «épingle à cheveux». De plus, l’étude du transcriptome a révélé 3 phases temporelles distinctes dans le cycle infectieux: précoce, intermédiaire et tardive. Les transcrits de capsides sont tous exprimés durant la phase tardive mais leur profil d’expression ne sont pas superposables dans le temps. Les données de transcriptomique ont révélées la présence de plusieurs glycosyltransférases chez Mimivirus, dans la phase tardive du cycle, concomitant avec la production de la protéine de capside. Les informations recueillies sur l'expression des gènes à différents temps post-infection ont contribué à la conception de protocoles pour la production des protéines de capsides (la protéine majeure de capside (MCP) et ses paralogues) dans de systèmes eucaryote. / Difficult to express proteins: a bottleneck for most biologists. I have chosen to use Acanthamoeba polyphaga Mimivirus as my study model. This giant dsDNA virus possesses post-translationally modified proteins, multi-protein structures and enzyme pathways never before seen in a virus, which makes it ideal for refractory studies. The ultimate goal of my thesis was to produce the capsid proteins of Mimivirus. The role of the capsid protein in the assembly of the viral particle, its infectivity, and molecular features are of great importance. To go from gene to protein, I participated in the comprehension of what governs the post-transcriptional termination in Mimivirus and equally participated in the global analysis of the transcriptome during the infectious cycle of Acanthamoeba by Mimivirus. We have shown that the Mimivirus transcripts are systematically polyadenylated in the regions forming a stem-loop secondary structure; even when a canonical poyadenylation signal is absent We concluded that Mimivirus polyadenylation obeys a strict “Hairpin rule”. Moreover, the transcriptomic study revealed three distinct temporal phases: early, intermediate and late. The capsid transcripts are all expressed during the late phase but their expression profiles are not superimposable. The transcriptomic data also revealed the presence of several Mimivirus glycosyltransferases in the late temporal phase, concomitant with the capsid proteins. The expression data gathered throughout my thesis has contributed to the rational design of a protein production experiment to produce the major capsid protein and its three paralogs in eukaryotic systems.

In eukaryotes, mRNA decay pathways are important for cellular response to various physiological conditions and also function in co-translational quality control systems that target translationally aberrant mRNAs for degradation. My work on identification and characterization of novel components and pathways of mRNA degradation and quality control in Saccharomyces cerevisiae is summarized below.I have identified Edc3p as a novel protein important for mRNA decay. Deletion of Edc3p leads to a defect in mRNA decay in strains deficient in decapping enzymes and, in combination with a block to the 3' to 5' decay pathway, causes exaggerated growth defects and synthetic lethality. An Edc3p-GFP fusion protein localizes in processing bodies, which are specialized cytoplasmic foci containing decapping proteins. Together, these observations indicate that Edc3p directly interacts with the decapping complex to stimulate the mRNA decapping rate.Quality control during mRNA translation is critical for regulation of gene expression. My work shows that yeast mRNAs with defects in translation elongation, due to strong translational pauses, are recognized and targeted for degradation via an endonucleolytic cleavage in a novel process referred to as No-Go Decay (NGD). The cellular mRNA decay machinery degrades the 5' and 3' cleavage products produced by NGD. NGD is translation-dependent, occurs in a range of mRNAs and can be induced by a variety of elongation pauses. These results indicate NGD may occur at some rate in response to any stalled ribosome.I also show that two highly conserved proteins, Dom34p and Hbs1p, homologous to the eukaryotic release factors eRF1 and eRF3 respectively, are required for NGD. Further characterization of the No-Go decay pathway indicates that Dom34p function during NGD is conserved across species. Identification of RPS30, a small ribosomal protein as a trans-acting factor during NGD suggests that the ribosome may have a novel role during NGD. Other experiments indicate that the No-Go decay pathway may cross talk with the unfolded protein response pathway. The identification of No-Go decay as a novel quality control pathway during translation elongation supports the existence of a global cellular mechanism for maintenance of translational quality control.

Regulation of gene expression is essential for many biological processes. Binding of transcription factors to DNA is a key regulatory step in the control of gene expression. It is commonly observed that DNA sequences with high affinity for transcription factors occur more frequently in the genome than the instances of genes bound or regulated by these factors. However, the mechanism by which transcription factors selectively identify and regulate these genes was unclear. I utilized the transcriptional control of the phosphate-responsive signaling pathway (PHO) in Saccharomyces cerevisiae as a model system to address this problem.

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