Les transposases sont des enzymes qui reconnaissent des séquences spécifiques sur l'ADN aux bornes des transposons (éléments à transposer) et catalysent des réactions de coupure et de transfert de brins. La mobilité des transposons entraîne la plasticité des génomes, et dans certains cas, l'exaptation de gènes de transposons contribue à l'émergence de nouvelles fonctions cellulaires. La paramécie est un modèle eucaryote unicellulaire extraordinaire pour étudier le rôle des transposases domestiquées de PiggyBac (nommées PiggyMac et PiggyMac-like) dans le réarrangement programmé de son génome. Ces dernières contribuent à l'assemblage de son génome somatique au cours du cycle sexuel. Les principaux axes de ce projet se sont centrés sur l’étude structurale de la transposase PiggyBac (issue de Trichoplusia ni) et une étude d’interaction avec des séquences particulières de son transposon. Ainsi qu’une étude structurale de la transposase domestiquée PiggyMac chez la paramécie.La première structure obtenue (domaine riche en cystéine de la transposase PiggyBac) montre une structuration en doigt de zinc de type PHD-RING. Il a été démontré in vivo et in vitro l’importance du domaine riche en cystéines (CRD) de PiggyBac pour une activité d’excision et d’intégration du transposon PiggyBac. Nous avons pu mettre en évidence que le CRD de PiggyBac cible des séquences spécifiques d’ADN qui sont localisées dans les séquences TIR (Terminal Inverted Repeat) gauche et droite du transposon. Grâce aux résultats issus de la RMN, des modèles de complexes protéine-ADN ont pu être établis.Concernant PiggyMac (transposase PiggyBac domestiquée), son domaine riche en cystéines et histidines a pu être produit doublement marqué (15N et 13C) dans E.coli. Des études structurales en RMN et l'utilisation d'un programme de modélisation moléculaire CYANA ont permis d’accéder à la structure tridimensionnelle de ce domaine.Nous montrons que celui-ci se replie en doigt de zinc entrelacé qui lie deux ions zinc avec un total de huit histidines et cystéines (résultat en cours de publication). La configuration de ce doigt de zinc est différente de celui de PiggyBac et même nouveau dans la littérature concernant ce peptide. Cette étude a permis de mettre en évidence un nouveau type de doigt de zinc. / Transposases are enzymes that recognize specific DNA sequences across transposons (elements to transpose) and catalyze cleavage and transfer reactions strands. The mobility of transposons causes the genome plasticity, and in some cases, the transposon gene exaptation contributes to the emergence of new cellular functions. Paramecium is an extraordinary unicellular eukaryotic model to study the role of transposases domesticated PiggyBac (named PiggyMac and PiggyMac-like) in the programmed rearrangement of its genome. These contribute to the assembly of the somatic genome during sexual cycle. The main axes of this project have focused on the structural study of the transposase PiggyBac (derived from Trichoplusia ni) and an interaction study with particular sequences of the transposon. And a structural study of the transposase domesticated PiggyMac in Paramecium.The first structure obtained (cysteine-rich domain of the transposase PiggyBac) shows a structure in PHD-RING-type zinc finger. It has been demonstrated in vivo and in vitro the importance of the cysteine-rich domain (CRD) of PiggyBac for excision activity and integration of the transposon PiggyBac. We were able to show that the CRD PiggyBac target specific DNA sequences that are located in the TIR sequences (Inverted Terminal Repeat) left and right of the transposon. Thanks to the NMR results, protein-DNA complexes models were established.Regarding PiggyMac (PiggyBac domesticated transposase), its cysteine ​​and histidine rich domain has been doubly labeled product (15N and 13C) in E. coli. NMR structural studies and the use of a CYANA molecular modeling program allowed to access the three-dimensional structure of this domain.We show that it folds into interlaced zinc finger that binds two zinc ions with a total of eight histidine and cysteine ​​(results being published). The configuration of this zinc finger is different from that of PiggyBac and even new in the literature concerning this peptide. The study highlighted a new type of zinc finger.

Although all cells in a multicellular organism contain the same set of genes, the spatiotemporal expression of these genes needs to be dynamically regulated for morphogenesis and life cycle transitions to take place. Polycomb-group (PcG) proteins are evolutionarily-conserved epigenetic regulators that function – via epigenetic marks such as H3K27me3 and modifications to chromatin structure – to maintain the repression of developmentally-important genes so that these genes are only expressed in the appropriate cells at the appropriate times. This repressive activity of the PcG is antagonised by the trithorax-group (trxG) of proteins. Although they maintain specific patterns of gene repression, PcG proteins are ubiquitously expressed. How their activity is regulated is largely unknown. To identify such regulatory pathways, a genetic screen for modifiers of PcG activity in Arabidopsis was carried out previously using the PcG mutant curly leaf (clf), which has moderately-severe developmental defects due to the ectopic or untimely expression of developmental regulators such as floral homeotic genes and the important flowering time regulator FLOWERING LOCUS T (FT). I characterised three novel potential regulators identified in this genetic screen: the chromatin-associated protein AT-HOOK MOTIF NUCLEAR LOCALISED PROTEIN 22 (AHL22), the 26S proteasome and the novel trithorax-group members ANTAGONIST OF LHP1 1 and 2 (ALP1 and ALP2). I found that the dominant sop-11D mutation is caused by over expression of AHL22 which suppresses the phenotype of clf by reducing FT expression. However, genetic analysis suggests that AHL22 may act in a parallel pathway to the PcG. I showed that mutations affecting diverse subunits of the 26S proteasome reduce the mis-expression of CLF targets and suppress the phenotypes of clf although they do not restore H3K27me3 levels at these targets. Pharmacological inhibition of the proteasome also alleviated the mis-expression of target genes found in clf mutants. Analysis of PcG protein levels following proteasome inhibition suggests that the 26S proteasome antagonises the PcG by degrading the key PcG member EMBRYONIC FLOWER 1 (EMF1), which is likely to be important for implementing target gene repression. Surprisingly, my proteomic analysis showed that the novel trxG members ALP1 and ALP2 are accessory components of a core PcG complex – the Polycomb Repressive Complex 2 (PRC2) – in vivo, suggesting that that ALP1 and ALP2 may antagonise PcG repression by preventing the association of core PRC2 components with accessory components EMF1, LIKE HETEROCHROMATIN PROTEIN 1 and the PHD finger proteins VERNALISATION5 and VIN3-LIKE 1. My results reveal a previously unknown role for 26S proteasomal degradation in the regulation of PcG activity during vegetative development and identify novel in vivo associators of the core PRC2 and point to their role in modulating PcG activity. These results thereby increase our understanding of how the PcG is regulated and serve as a starting point to discover how specificity is given to the PcG mediated repression, either by targeted degradation of EMF1 by various E3 ligases or by different combinations of PRC2 associators.

Made available in DSpace on 2015-03-26T13:51:56Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1477917 bytes, checksum: 4ab8f5cc2dd481acd067b02bd7abf32d (MD5) Previous issue date: 2012-01-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Colletotrichum is one of the most important genera of plant-pathogenic fungi in the world. The pathogenic species of this genus have hemibiotrophic lifestyle and cause diseases in several economically significant crops. Besides the economic importance, Colletotrichum has great significance as a model system for studying the molecular and cellular bases of fungal pathogenicity. The species C. graminicola, causal agent of corn anthracnose (Zea mays), has rare sexual stage and was the first species of the genus to have its genome completely sequenced. The transposable elements are ubiquitous and constitute a source of new mutations, being an important source of genetic variability. These elements are divided into two classes according to the presence or absence of an RNA intermediate in transposition. Elements of class I transpose via RNA intermediate, while class II elements transpose directly as DNA. The transposable elements can be applied as mutagenic agents aimed at the identification and labeling of genes and in phylogenetic and population studies. Given the importance of transposable elements in the generation of genetic variability and its applications in research, the aim of this study was to identify and characterize the class II transposable elements in the genome of C. graminicola. For this purpose, we used a bioinformatic approach combined with experimental activities. We identified 132 complete sequences of transposable elements in the sequenced genome of C. graminicola, which represent a significant proportion of the genome (0.47%). The elements were classified into six families according to similarity, all elements have characteristics of Tc1-mariner superfamily. Although some of these elements possess putative transposases with conserved DDE domain, all are interrupted by multiple stop codons. None of the elements identified has all the necessary features to be considered an active element. In silico analysis revealed evidence that these sequences are mutated by RIP (repeat point induced mutation) mechanism. TCg1 element was amplified by PCR from a Brazilian isolate and has imperfect terminal inverted repeats and the putative transposase sequence has three conserved domains characteristic of transposases: DDE, CENPB and HTH. However, this sequence is interrupted by stop codons and lacks the initiation codon and termination codon, therefore, is probably inactive. The genomic DNA from 49 different isolates were analyzed by hybridization with a probe derived from the inner region of TCg1 and different profiles were identified. The strategy allowed the efficient identification of a variety of Tc1-Mariner transposable elements degenerated by mutations characteristics of RIP in C. graminicola. It is unlikely that any of the identified elements is autonomous, however, these elements must have an important role in the genetic variability of this fungus. The TCg1 element is present in the genomes of different isolates of C. graminicola and has the potential to be used as a molecular marker in population analyzes. / Colletotrichum é um dos gêneros mais importantes de fungos fitopatogênicos em todo o mundo. As espécies fitopatogênicas desse gênero apresentam ciclo de vida hemibiotrófico e causam doenças em diversas culturas economicamente importantes. Além da importância econômica, Colletotrichum possui grande relevância como um sistema modelo para o estudo das bases celulares e moleculares da patogenicidade fúngica. A espécie Colletotrichum graminicola, agente causal da antracnose do milho (Zea mays), possui ciclo sexual raro e foi a primeira espécie do gênero a ter o seu genoma completamente sequenciado. Os elementos transponíveis são ubíquos e constituem uma fonte de novas mutações, sendo, portanto, uma importante fonte de variabilidade genética. Esses elementos são divididos em duas classes de acordo com a presença ou ausência de um intermediário de RNA na transposição. Os elementos da classe I se transpõem via intermediário de RNA, enquanto os elementos da classe II se transpõem diretamente como DNA. Os elementos transponíveis podem ser utilizados como agentes mutagênicos visando à identificação e etiquetagem de genes e em estudos filogenéticos e populacionais. Tendo em vista a importância dos elementos transponíveis na geração de variabilidade genética e as suas aplicações na pesquisa, o objetivo deste trabalho foi identificar e caracterizar elementos transponíveis da classe II no genoma de C. graminicola. Para tanto, foi utilizada uma abordagem de bioinformática (análises in silico) aliada às atividades experimentais. Foram identificadas 133 sequências completas de elementos transponíveis no genoma sequenciado de C. graminicola, que representam uma proporção relevante do genoma (0,47%). Os elementos foram classificados em 6 famílias de acordo com a identidade e apresentam características da superfamília Tc1-Mariner. Apesar de algumas transposases putativas codificadas por esses elementos possuírem domínio DDE conservado, todas estão interrompidas por vários códons de parada. Nenhum elemento identificado possui todas as características necessárias para um elemento autônomo. A análise in silico revelou evidências de mutações geradas pelo mecanismo de RIP (Mutação de ponto induzida por repetição). O elemento TCg1, amplificado por PCR a partir de um isolado brasileiro de C. graminicola, possui extremidades repetidas invertidas imperfeitas e a sequência putativa da transposase apresenta os três domínios característicos conservados: DDE, HTH e CENPB. Entretanto, essa sequência está interrompida por códons de parada e não foram localizados os códons de iniciação e de terminação, sendo, portanto, provavelmente inativa. O DNA genômico de 49 diferentes isolados foi analisado por hibridização com uma sequência derivada da região interna de TCg1 e apresentaram diferentes perfis. A estratégia utilizada permitiu uma identificação eficiente de uma variedade de elementos transponíveis Tc1-Mariner degenerados por mutações características de RIP em C. graminicola. É improvável que algum dos elementos identificados seja autônomo, entretanto, esses elementos devem possuir um importante papel na variabilidade genética desse fungo. O elemento TCg1 está presente no genoma de diferentes isolados de C. graminicola e possui potencial para ser utilizado como marcador molecular em análises populacionais.

La résistance aux antibiotiques est un problème majeur de santé publique. Les éléments génétiques mobiles, comme les séquences d’insertion (IS), jouent un rôle important dans la dissémination des gènes de résistance, notamment chez les entérobactéries, pathogènes majeurs chez l’Homme. Nous avons identifié une IS atypique, l’IS1294b (1713 pb) en amont du gène de la céphalosporinase blaCMY-2 présent sur le plasmide conjugatif (p2735) d’une souche d’origine clinique de Klebsiella pneumoniae résistante à haut niveau à la ceftazidime (céphalosporine de 3ième génération). L’IS1294b appartient à la famille des IS91-like qui transpose probablement selon le mode de la réplication du cercle roulant. Nous avons montré expérimentalement que l’IS1294b était capable de mobiliser le gène blaCMY-2 d'un plasmide à un autre, via le mécanisme de "one-ended transposition". Cette transposition, se produisant avec une fréquence comprise entre 1,7 et 14,0%, et implique la non-reconnaissance de l’une de ses extrémités (terIS). Nous avons développé un test chez Escherichia coli et étudié l’effet des mutations introduites dans l’IS1294b sur la fréquence de transposition in vivo. Nous avons identifié 8 nucléotides critiques à l’extrémité oriIS, et un motif de liaison à l’ADN probable (6 cystéines et 1 histidine importantes) dans le domaine N-terminal de la transposase. La délétion de la région 1 à 24 de l’extrémité terIS pourrait favoriser efficacement le mécanisme de « one-ended transposition ». Des résidus tyrosines (Y254 et Y258) et histidines (H164, H166 et H153) sont indispensables à l’activité de cette transposase Y2 et conforte son appartenance à la superfamille des protéines HUH. La purification, en cours, de la transposase (fusionnée à la thioredoxine pour la solubiliser) permettra l’étude ultérieure de son activité in vitro. Notre étude constitue une première description expérimentale de la mobilisation d'un gène d’une ß-lactamase par un élément appartenant à la famille des IS91 et ouvre des voies dans la compréhension du mécanisme de transposition de ces éléments génétiques mobiles. / Antibiotic resistance is a major public health issue. Mobile genetic elements, such as the insertion sequences (IS), play an important role in the dissemination of antibiotic resistance genes mainly in enterobacteria, major human pathogens. We identified an atypical IS, IS1294b (1713 bp), upstream the cephalosporinase gene, blaCMY-2. They were located on the conjugative plasmid (p2735) from a clinical strain of Klebsiella pneumoniae, highly resistant to ceftazidime (third generation cephalosporin). IS1294b belongs to the IS91-like family which probably transposes according to the mode of rolling circle replication. We have shown experimentally that IS1294b was able to mobilize the blaCMY-2 gene from a plasmid to another, through the mechanism of "one-ended transposition". This mobilization involving the non-recognition of the terIS end occurred with a percentage ranging between 1.7 and 14%. We developed a transposition test in Escherichia coli and studied the effect of mutations introduced into the IS1294b on in vivo transposition frequency. We identified 8 critical nucleotides at the oriIS end, and a probable DNA binding motif (6 cysteines and one histidine are essential) in the N-terminal domain of the transposase. The deletion of the region 1-24 at the terIS end could enhance effectively the mechanism of "one-ended transposition." Tyrosines residues (Y254 and Y258) and histidines (H164, H166 and H153) are essential to the activity of the transposase Y2 and reinforce its belonging to the HUH protein superfamily. Purification of the transposase (fused to the thiroredoxine for solubilization) is in progress and will allow further study of its in vitro activity. Our study is the first experimental description of the mobilization of a beta-lactamase gene by an element belonging to the IS91 family, and could initiate the understanding of the mechanism of transposition of these mobile genetic elements.

To explore the similarities and differences of regulatory circuits among budding yeasts, we characterized the role of unscheduled meiotic gene expression 6 (UME6) and a novel mating type switching pathway in Kluyveromyces lactis. We found that Ume6 was required for transcriptional silencing of the cryptic mating-type loci HMLα and HMRa. Ume6 acted directly at these loci by binding to the cis-regulatory silencers. Ume6 also served as a block to polyploidy and was required for repression of three meiotic genes, independently of the Rpd3 and Sin3 corepressors. Mating type switching from MATα to MATa required the α3 protein. The α3 protein was similar to transposases of the mutator like elements (MULEs). Mutational analysis showed that the DDE-motif in α3, which is conserved in MULEs was necessary for switching. During switching α3 mobilizes from the genome in the form of a DNA circle. The sequences encompassing the α3 gene circle junctions in the MATα locus were essential for switching from MATα to MATa. Switching also required a DNA binding protein, Mating type switch 1 (Mts1), whose binding sites in MATα were important. Expression of Mts1 was repressed in MATa/MATα diploids and by nutrients, limiting switching to haploids in low nutrient conditions. In a genetic selection for strains with increased switching rates we found a mutation in the RAS1 gene. By measuring the levels of the MTS1 mRNA and switching rates in ras1, pde2 and msn2 mutant strains we show that mating type switching in K. lactis was regulated by the RAS/cAMP pathway and the transcription factor Msn2. ras1 mutants contained 20-fold higher levels of MTS1 mRNA compared to wild type whereas pde2 and msn2 expressed less MTS1 mRNA and had decreased switching rates. Furthermore we found that MTS1 contained several potential Msn2 binding sites upstream of its ORF. We suggest that these observations explain the nutrient regulation of switching. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.

Bacteriophage Mu exhibits low specificity for the 5 bp sequence it selects as its transposition target, but shows regional biases in its insertion choices. For example, Mu prefers AT-rich DNA in vitro, exhibits a 1000-fold bias in target preference within the E. coli chromosome, and avoids targets carrying Mu end sequences. The Mu transposase is responsible for recognition of the 5 bp target consensus, but depends on the accessory protein MuB for efficient target capture. MuB preferentially binds to AT-rich DNA, explaining this particular regional preference. We have uncovered opposing roles for MuB in target capture and integration. We show while MuB-bound AT-rich DNA is favored for integration, the bound DNA itself is refractory, and that transposition occurs adjacent to, but not within the bound region. We show that this property of MuB is likely responsible for immunity of Mu from self-integration, since MuB was found to be strongly bound within the Mu genome. Genome-wide analysis of MuB binding on the E. coli chromosome showed that Mu target preference is positively related to MuB binding profile, and that MuB binding is insulated by the nucleoid-associated protein Fis but not by transcription events. Since Fis binding to the chromosome responds to the frequency of A-tracts, a chromosome domain structure signal, Mu transposition must also respond to chromosome domain signals. Work in this dissertation has provided a new understanding of how MuB influences and controls Mu target choice, and of reciprocal interactions between a bacterial chromosome and a transposable element. / text

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-14T11:57:27Z No. of bitstreams: 1 resumo.pdf: 17456 bytes, checksum: 1d66a7d5cc0063d744a4b711bc90ee10 (MD5) / Made available in DSpace on 2017-06-14T11:57:27Z (GMT). No. of bitstreams: 1 resumo.pdf: 17456 bytes, checksum: 1d66a7d5cc0063d744a4b711bc90ee10 (MD5) Previous issue date: 2004-05-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Foi feita a caracterização de um mutante espontâneo do Lactobacillus delbrueckii UFV H2b20 que mostrou-se desprovido da capacidade de imunoestimulação e também a investigação do possível papel da seqüência de inserção ISLdH2b20 nessa mutação. A confirmação da identidade genética de L. delbrueckii UFV H2b20 com a do mutante desprovido da capacidade de imunoestimular o hospedeiro foi realizada por análise dos perfis de PCR-DGGE. Ambas bactérias apresentaram na microscopia eletrônica de transmissão característica peculiar do H2b20, a de formar bastonete alongado quando na fase estacionária. As células do mutante mostraram-se com diâmetro maior do que as da cultura do L. delbrueckii UFV H2b20, porém as velocidades especificas máximas de crescimento (μ max ) foram iguais. As células do mutante sofrem lise mais rápida do que as da cultura original selvagem quando em presença de baixas concentrações de mutanolisina, no entanto, o perfil de lise é idêntico em presença de lisozima. A hidrofobicidade de superfície das células, determinada pelo teste de MATH, é maior nas células da cultura original do L. delbrueckii UFV H2b20. Os perfis de proteínas de superfície não se distinguem quanto ao número e posição das bandas em SDS-PAGE, contudo a banda referente à proteína de aproximadamente 36kDa apresentou mais proteína nos extratos do tipo selvagem do que a do mutante. O mutante apresentou uma maior quantidade de proteínas em quase todas as outras bandas, com destaque para duas bandas, 60 kDa e 75 kDa. O estudo de distribuição da ISLdH2b20 nos isolados selvagem e mutante, feito por análise de Southern, mostrou mesmo perfil de distribuição dessa seqüência de inserção nos dois solados. Os resultados indicam a possível participação de uma proteína de 36 kDa no processo de imunoestimulação do H2b20, possivelmente da camada S, e que a instabilidade desse, quanto à capacidade de imunoestimulação, não tem a participação direta da seqüência de inserção ISLdH2b20 em particular. / A mutant of L. delbrueckii UFV H2b20, without the capacity of immunestimulation, was characterized and the possible role of the insertional sequence ISLdH2b20 in this mutation also. PCR-DGGE confirmed its genetic identity as L. delbrueckii UFV H2b20. Transmission electron microscopy demonstrated the characteristic morphology of the cells in the stationary phase, when they appeared as long rods with 0,653μm diameter, for the wild type, and 0,735μm, for the mutant. Maximum specific growth rate (μ max ) was similar for both cultures. The effect of lysozyme on cells of H2b20 and mutants was the same; however, mutanolysin had a more rapid lytic effect on mutant cells, when in lower concentrations. The superficial hydrophobicity of the cells, determined by microbial adhesion to hexadecane (MATH) is higher in wild type cells. The profiles protein of superficial proteins, by SDS-PAGE, are similar when the position and number of bands are considered, however, the band represented by one protein with approximately 36kDa showed more protein in the wild type extract than in the mutant extract. The mutant presented more proteins in almost all of the other bands, especially in the two bands representing proteins with 60kDa and 75kDa. The consolidated results point to a possible role for the 36kDa protein in the immunestimulation capacity of the wild type cells. This is possible the S-layer protein of L. delbrueckii UFV H2b20. Southern analysis of the wild type and the mutant’s DNAs indicated no difference in the distribution of the several copies of ISLdH2b20 between the two strains. This leads to the conclusion that the mutation, which introduced to loss of the immunestimulation capacity, is not the result transposition of this putative element.

Tn7 and Tn7-like transposons are complex elements found in disparate environments and are responsible for mobilizing a wide variety of genes and forming pathogenicity/fitness islands. They are novel in their ability to recognize both a single site in the chromosome and specifically target transposition into mobile plasmids via dedicated TnsD and TnsE targeting proteins. TnsE recognizes mobile plasmids through an association with the processivity clamp and a 3′ recessed DNA end during conjugal replication. However, the mechanism for the specific recognition of 3′ recessed DNA ends remains unclear. Structural analyses of the C-terminal domain of TnsE identified a novel protein fold including a central V-shaped loop that toggles between two distinct conformations. The structure of a robust TnsE gain-of-function variant has this loop locked in a single conformation, suggesting that conformational flexibility regulates TnsE activity. Structure-based analysis of a series of TnsE variants relates transposition to DNA binding stability. Follow up studies of full length TnsE bound to DNA are in progress. / Thesis / Master of Science (MSc)

No description available.