Il gene-targeting in colture cellulari associato alla tecnica del trasferimento nucleare oggi rappresenta il sistema d'elezione nella creazione di animali transgenici. Purtroppo la ricombinazione omologa (HR) è poco efficiente soprattutto in cellule somatiche. La positive-negative selection (PNS) è la tecnica di arricchimento usata per geni non attivamente trascritti nel tipo cellulare utilizzato. In questo lavoro abbiamo scelto come locus bersaglio la b-lattoglobulina bovina e testato tre nuove cassette di selezione negativa, che non codificando per antibiotico-resistenze, determinano condizioni di coltura meno tossiche. / Gene-targeting of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. Nevertheless homologous recombination (HR) is a low frequency event in mammalian cells, above all in somatic cells. Positive-negative selection (PNS) is the enrichment strategy to target genes that are not actively transcribed in the cell type of choice. In this work we chose to target the bovine b-lactoglobulin gene and we tested three new negative selection cassettes in bovine fibroblasts. Such new targeting vectors allow a single selective drug employ and produce less toxic culture conditions.

The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3’ single-stranded DNA for recombinase loading and strand exchange. In thermophilic Archaea, the mre11 and rad50 genes cluster in an operon with genes encoding a bidirectional DNA helicase, HerA, and a 5’ to 3’ exonuclease, NurA, suggesting these four enzymes function in a common pathway. I show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5’ strand at a DNA end under physiological conditions in vitro where HerA and NurA alone do not show detectable activity. Furthermore, I demonstrate that HerA and NurA physically interact, and this interaction stimulates both helicase and nuclease activities. The products of HerA/NurA long-range resection are oligonucleotide products and HerA/NurA activity demonstrates both sequence specificity and a preference to cut at a specific distance from the DNA end. I demonstrate a novel activity of Mre11/Rad50 to make an endonucleolytic cut on the 5’ strand, which is consistent with a role for the Mre11 nuclease in the removal of 5’ protein conjugates. I also show that Mre11/Rad50 stimulates HerA/NurA-mediated resection through two different mechanisms. The first involves an initial Mre11 nucleolytic processing event of the DNA to generate a 3’ ssDNA overhang, which is then resected by HerA/NurA in the absence of Mre11/Rad50. The second mechanism likely involves local unwinding of the DNA end in a process dependent on Rad50 ATPase activity. I propose that this unwinding step facilitates binding of HerA/NurA to the DNA end and efficient resection of the break. Furthermore, the binding affinity of NurA for 3’ overhang and unwound DNA end substrates partially explains the efficiency of the two resection mechanisms. Lastly, 3’ single-stranded DNA generated by these enzymes can be used by the Archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in Archaea, and may serve as a model for DSB processing in eukaryotes. / text

Horizontal gene transfer (HGT) is probably the most important mechanism for functional novelty and adaption in bacteria. However, a robust understanding of the rates of HGT for most bacterial species and the influence of the ecological settings on the rates remain elusive. Four whole-genome comparative studies of free-living bacteria will be described that integrated physiological and ecological data with novel detection bioinformatic pipelines to elucidate the magnitude of HGT at three distinct levels of genetic relatedness: i) the species level, where overlapping ecological niche among co-occurring bacteria in the water column of the Baltic Sea has caused HGT to have been so rampant that it has served as the force of species cohesion; ii) the genus level, where HGT appeared to predominantly mobilize a limited number of genes with ecological/selective advantage (e.g., antibiotic resistance genes) among distinct pathogenic Campylobacter species and hence, did not lead to species convergence; and iii) the phylum level, where HGT was found to be, in general, less frequent than the genus level but, over evolutionary time, has assembled a large part of the metabolic functions of natural microbial communities, especially within organic matter rich, anaerobic habitats. In conclusion, this work advances the methods to link ecological relationships with HGT and suggests that HGT among very divergent organisms may have been more frequent than previously thought and led to successful adaptation.

<p>The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene.</p>

L'intégrité de l'information génétique est essentielle aux fonctions cellulaires et pour éviter l'instabilité génomique, qui est une des caractéristique du cancer. Suite à des cassures double brin (Double Strand Breaks; DSBs), la voie de signalisation de réponse aux dommages de l'ADN est activée dans la cellule qui comprend deux sous voies de signalisation : la jonction d'extrémités non-homologues et la recombinaison homologue. Le complexe TREX-2 associé au pore nucléaire est impliqué dans l'export des ARNm. Chez la levure, TREX-2 est impliqué dans le maintien de la stabilité génomique. Nous nous sommes intéressés au rôle de TREX-2 dans la réparation de DSBs dans les cellules humaines. La déplétion du complexe TREX-2 entraine une réparation de l'ADN par recombinaison homologue insuffisante. De plus, nos résultats démontrent que la protection contre les dommages de l'ADN par TREX-2 dépend aussi de l'équilibre entre H2B an H2Bub1 contrôlé par le module de deubiquitination de SAGA. / The maintenance of proper genetic information is essential to avoid genomic instability, which is a hallmark of cancer. In response to Double Strand Breaks (DSBs), cells initiate the DNA Damage Response (DDR), that acts through two main sub-pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). The nuclear pore-associated TREX-2 complex is involved in mRNA export and has been implicated, in yeast, in genome stability maintenance. Here we investigated the role of TREX-2 in DSB repair in human cells. We find that loss of the scaffold subunit of TREX-2 (GANP) results in DNA repair deficiency by HR. Moreover, we showed that the mechanism through which TREX-2 protects human cells from DNA damage is dependent on an interplay with the co-activator complex SAGA that regulates H2Bub1 histone mark. Our results demonstrate a functional cross-talk between human TREX-2 and the SAGA deubiquitination activity that is important to ensure correct DSB repair during HR.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-16T21:26:49Z No. of bitstreams: 1 Elsio Augusto Wunder Júnior Patogênese da leptospirose...pdf: 2609622 bytes, checksum: aa34c959355bc105a6e849e74258cf78 (MD5) / Made available in DSpace on 2012-07-16T21:26:49Z (GMT). No. of bitstreams: 1 Elsio Augusto Wunder Júnior Patogênese da leptospirose...pdf: 2609622 bytes, checksum: aa34c959355bc105a6e849e74258cf78 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A leptospirose é uma zoonose de importância global e um importante problema de saúde pública principalmente em países em desenvolvimento. É causada por bactérias do gênero Leptospira, uma espiroqueta móvel e de alta morbidade capaz de se disseminar nos tecidos e causar doença crônica em animais hospedeiros. Uma barreira importante para o controle e prevenção da doença tem sido o pouco conhecimento da patogênese do agente, em parte pela falta de ferramentas disponíveis e eficazes de manipulação genética. Um dos objetivos desse estudo foi caracterizar duas cepas mutantes de Leptospira interrogans. A interrupção do gene lipl32, que codifica para a proteína LipL32, a mais abundante proteína no gênero Leptospira e expressa somente na superfície das leptospiras patogênicas, foi realizada através da inserção do transposon Himar1 no sorovar Manilae. A cepa mutante não apresentou nenhuma diferença de crescimento ou de aderência em componentes da matriz celular, comparada com a cepa parental. O mutante foi capaz de produzir doença aguda no modelo animal de hamster e causar colonização crônica no modelo animal de rato, mostrando que LipL32 não possui um papel nestes modelos de infecção. A interrupção do gene ligB foi realizada com a utilização, pela primeira vez em leptospiras patogênicas, da técnica de recombinação homóloga por mutagênese dirigida, onde o gene que codifica para a proteína LigB teve uma parte substituída por um cassete de resistência de espectinomicina (Spcr). Essa proteína, identificada como um possível fator de virulência, reconhecida pelo soro de pacientes infectados e importante para a aderência em componentes da matriz celular, mostrou não ser importante para a infecção aguda ou crônica, quando testada frente aos modelos animais, além de não ser necessária para a aderência em cultura de células. Outro objetivo desse trabalho foi estudar a cinética de disseminação da Leptospira interrogans no modelo animal de hamster, utilizando uma dose alta (108 leptospiras) e baixa (250 leptospiras) de inóculo, além de diferentes rotas de infecção. Nossos resultados demonstraram que leptospiras se disseminam rapidamente em todos os tecidos 01 hora após a infecção com uma alta dose de inóculo e que possivelmente a carga do agente nos tecidos é mais importante para a patogênese do que a sua habilidade para a disseminação. Também demonstramos que a motilidade não é essencial para disseminação, mas pode ser essencial para a carga nos tecidos e letalidade. / Leptospirosis is a worldwide zoonosis and a major public health problem especially important in developing countries. Caused by bacteria of Leptospira genus, a motile lifethreatening spirochete which is able to disseminate to tissues and causes chronic carriage in animal hosts. A significant barrier to the control and prevention of leptospirosis has been the limited understanding of its pathogenesis, due in part to the lack of tools available for the genetic manipulation of this pathogen. One of the purposes of this study was to characterize two mutant strains of Leptospira interrogans. Interruption of lipL32 gene, encoding LipL32, the most abundant protein of pathogenic leptospires and its major outermembrane lipoprotein, was achieved in serovar Manilae using transposon mutagenesis with Himar1. The mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. The mutant was able to cause acute severe disease manifestations in the hamster model and chronic colonization in the rat model, showing that LipL32 doesn’t play a role in neither of those models of infection. Interruption of ligB gene was achieved using the homologous recombination by target mutagenesis for the first time in pathogenic leptospires and a spectinomycin resistance (Spcr) gene replaced a portion of the ligB coding sequence. This Lig protein, previously identified as a putative virulence factor, recognized by the sera of infected patients and important for the adherence in extracellular matrix components, showed not to be important for acute or chronic infection when tested with animal models and it’s not required to mediate bacterial adherence to cultured cells. Another purpose of this study was to determine and analyze the kinetics of dissemination of Leptospira interrogans in the hamster model, using a high (108 leptospires) and low (250 leptospires) dose of inoculum and different routes of infection. Our results demonstrated that leptospires can rapidly disseminate through all tissues after 1 hour post-challenge with a high inoculum dose and that perhaps the load of the agent in target tissues is more important for pathogenesis than its ability for dissemination. We also demonstrate that motility is not essential for dissemination but can be essential for tissue load and lethality.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The first study characterized the expression of MAS receptor and key enzymes for Ang-(1-7) production, such as, ACE2, NEP and PEP during follicular development. Furthermore, the regulation of local Ang1-7 system was evaluated after the intrafollicular injection of fulvestrant (an estradiolreceptor inhibitor) in the dominant follicle. Cows were ovariectomized when the size between the largest (F1) and the second largest follicle (F2) was not statistically different (Day 2), slightly different (Day 3), or markedly different (Day 4). The mRNA abundance of genes encoding MAS receptor, ACE2, NEP and PEP was evaluated in the follicular cells from F1 and F2. The mRNA expression of MAS receptor was upregulated in the granulosa cells of F2 after the establishment of follicular deviation (Day 4), while PEP mRNA increased during (Day 3) and after (Day 4) the deviation process. However, the mRNA expression of ACE2 was upregulated in the granulosa cells of F1 during and after the deviation process. The mRNA expression of NEP was not regulated in F1 and F2. The MAS receptor was immunolocated in the granulosa and theca cells of F1 and F2 during follicular deviation. Moreover, MAS receptor gene expression increased when the F1 was treated with the estrogen receptor-antagonist in vivo. In conclusion, the expression profile of MAS receptor, ACE2, NEP and PEP in dominant and subordinate follicles indicated that Ang-(1-7) play a role in the regulation of the follicular dominance in cattle. A second study was performed to investigate the expression of genes that control homologous recombination (HR; 53BP1, ATM, RAD50, RAD51, RAD52, BRCA1, BRCA2 and NBS1), and non-homologous end-joining (NHEJ; KU70, KU80 and DNAPK), DNArepair pathways in bovine embryos with high, intermediate or low developmental competence. We also evaluated whether bovine embryos can respond to DNA double-stranded breaks (DSBs) induced by ultraviolet (UV) irradiation by regulating the expression of genes involved in the HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro fertilization and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation (EGA). All studied genes were expressed before, during and after the EGA period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before EGA in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-fertilization in bovine embryos with DSBs induced by UV irradiation. In conclusion, important genes controlling HR and NHEJ repair pathways are expressed in bovine embryos before, during or after EGA. Lower developmental competence seems to be associated with a higher mRNA expression of 53BP1 and RAD52. Bovine embryos can response to UV-induced DSBs after the EGA but HR and NHEJ repair pathways seem to be particularly regulated at the blastocyst stage. / O primeiro estudo caracterizou a expressão do receptor MAS e de enzimas responsáveis pela produção de Ang-(1-7), tais como, enzima conversora de angiotensina 2 (ACE2), endopeptidase neutra (NEP) e prolil endopeptidase (PEP) durante o desenvolvimento folicular. Além disso, a regulação local do sistema Ang-(1-7) foi avaliada após a injeção intrafolicular de fulvestrant (inibidor do receptor de estradiol) no folículo dominante. As vacas foram ovariectomizadas quando o tamanho entre o maior (F1) e o segundo maior folículo (F2) não era estatisticamente diferente (D2), ligeiramente (D3) ou marcadamente diferente (D4). A expressão de RNAm do receptor MAS, ACE2, NEP e PEP foi avaliada nas células foliculares do F1 e F2. O receptor MAS foi mais expresso nas células da granulosa do F2 após o estabelecimento da divergência folicular (D4), enquanto a expressão de PEP aumentou durante (D3) e após (D4) o processo de divergência. Entretanto, a expressão de ACE2 foi maior nas células da granulosa do F1 durante e após a divergência. A expressão de PEP não foi regulada no F1 e F2. O receptor MAS foi imunolocalizado nas células da teca e granulosa do F1 e F2 durante a divergência folicular. A expressão de RNAm do receptor MAS aumentou quando o F1 foi tratado com fulvestrant in vivo. Em conclusão, o perfil de expressão do receptor MAS, ACE2, NEP e PEP nos folículos dominante e subordinado indicam que a Ang-(1-7) apresenta uma função na regulação da dominância folicular em bovinos. Em um segundo estudo investigamos a expressão de genes que controlam o reparo do DNA através das vias de recombinação homóloga (HR; 53BP1, ATM, RAD50, RAD51, RAD52, BRCA1, BRCA2, NBS1) e união terminal não homóloga (NHEJ; KU70, KU80, DNAPK) em embriões bovinos com alta, média ou baixa competência de desenvolvimento. Foi também avaliado se embriões bovinos podem responder a quebra na fita dupla de DNA (DSBs), induzida por irradiação UV, através da regulação de genes envolvidos nas vias de reparo HR e NHEJ. Embriões com alta, média ou baixa competência de desenvolvimento foram selecionados pelo tempo de clivagem após a fertilização in vitro e foram removidos do cultivo antes (36 h), durante (72 h) ou após (96 h) o momento esperado para a ativação do genoma embrionário (AGE). Todos os genes foram expressos antes, durante e após a AGE independentemente da competência de desenvolvimento dos embriões. A expressão de 53BP1 e RAD52 foi maior antes da AGE em embriões com baixa competência de desenvolvimento. A expressão de 53BP1, RAD51 e KU70 foi mais baixa as 72 h e maior as 168 h pós fertilização em embriões com DSBs induzida por irradiação UV. Em conclusão, genes importantes para o controle das vias de reparo HR e NHEJ são expressos em embriões bovinos independentemente do tempo de cultivo ou da competência de desenvolvimento. A menor competência de desenvolvimento embrionário parece estar associada com maior expressão de 53BP1 e RAD52. Os embriões bovinos respondem a DSBs após a AGE mas as vias HR e NHEJ são reguladas principalmente no estágio de blastocisto.

Os micro-organismos são profícuas fontes de produtos naturais bioativos. Diversos fármacos de importância clínica são de origem microbiana, sendo que a maioria dos antibióticos usados clinicamente é produzida por actinobactérias, principalmente do gênero Streptomyces. A resistência a múltiplas drogas por microorganismos patogênicos e também pelas células tumorais leva à necessidade por novos fármacos antibacterianos e antitumorais. Actinobactérias endofíticas têm demonstrado grande potencial para a busca de produtos naturais bioativos. O presente trabalho relata o estudo químico de duas linhagens de actinobactérias endofíticas, Streptomyces sp. RTd 22 e Streptomyces sp RTd 31, isoladas das raízes de Tithonia diversifolia. As frações ativas nos ensaios biológicos foram fracionadas para a identificação dos compostos bioativos, sendo eles os antibióticos macrolídeos concanamicinas A (S31-1) e B (S31-2), anidro-agliconas das concanamicinas A (S31-3) e B (S31-4), todos produzidos por Streptomyces sp RTd31, e o ionóforo poliéter grisorixina (S22-2), produzido por Streptomyces sp. RTd22. Foi realizado o monitoramento da produção desses compostos bioativos por UPLC-MS através do modo SIM. As concanamicinas A e B tiveram um máximo de produção com 96h, já a grisorixina obteve um máximo com 192h. Outros compostos identificados por desreplicação dos extratos butanólicos de ambas as actinobactérias foram os sideróforos norcardamina (S31-7) e desoxi-nocardamina (S31-8), já o sideróforo desferrioxamina B (S31-9) foi identificado apenas nos extratos butanólicos de Streptomyces sp RTd31. Experimentos de variação do meio de cultivo e co-cultura com bactérias patogênicas foram empregados a fim de estimular a biossíntese de novos compostos, porém nenhum novo metabólito foi identificado. O sequenciamento genético da actinobactéria Streptomyces sp. RTd22 permitiu verificar a presença de vários clusters biossintéticos nesse micro-organismo através da análise feita pelo antiSMASH. Foi possível identificar o cluster da himastatina (S22-4) e da coeliquelina (S22-5), sendo que ambos os compostos não foram biossintetizados nas condições de cultivo utilizadas. O cluster biossintético da grisorixina foi determinado e o experimento de recombinação homóloga para a deleção do gene análogo a flavina mono-oxigenase da nigericina nigC foi realizado. Dois mutantes foram obtidos e um deles foi cultivado para a análise do perfil metabólico por espectrometria de massas. Não houve a produção da grisorixina nem do seu possível precursor pelo mutante, mas outros metabólitos foram produzidos / Microorganisms are prolific sources of bioactive natural products. Several clinically important drugs have microbial origin, and most of the therapeutically used antibiotics are produced by actinobacteria, mainly from the genus Streptomyces. The multidrug resistance observed in pathogenic microorganisms and tumor cells lead to the need for new antibacterial and antitumor drugs . Endophytic actinobacteria have shown great potential in the search for bioactive natural products. This work describes the chemical study of two endophytic actinobacteria strains: Streptomyces sp. RTd 22 and Streptomyces sp RTD 31, isolated from Tithonia diversifolia roots. Active fractions in biological assays were further fractionated for identifying the bioactive compounds, which are: the macrolide antibiotics concanamycins (S31-1) and B (S31-2), anhydrous aglycones of concanamycins A (S31-3) and B (S31-4), all four produced by Streptomyces sp. RTd31, and the ionophore polyether grisorixin (S22-2), produced by Streptomyces sp. RTd22. The production of these bioactive compounds was monitored by UPLC-MS via the SIM mode. Concanamycins A and B had maximum production at 96 h, and grisorixin at 192 h. Other compounds identified by the dereplication of buthanolic extracts of both actinobacteria were the siderophore norcardamine (S31-7) and deoxy-nocardamine (S31-8), the siderophores desferrioxamine B (S31-9) was identified only in buthanolic extracts of Streptomyces sp RTd31. Experiments varying media and co-culture were tested to stimulate the biosynthesis of novel compounds, but nothing new was identified. By genome sequencing of Streptomyces sp RTd22 and antiSMASH analysis it was possible to verify the presence of several biosynthetic clusters in the genome of this strain. It was possible to identify the biosynthetic clusters of himastatin (S22-4) and its analogous compound coelichelin (S22-5); however, these compounds were not biosynthesized in the culture conditions used. The grisorixin biosynthetic cluster was determined, and homologous recombination was performed for deleting the analogue gene of nigericin flavin monooxygenase nigCI. Two mutants were obtained, and one of them was cultured for analyzing its metabolic profile by mass spectrometry. There was no production of grisorixin or its possible precursor by the mutant, but others compounds were produced.

Os micoplasmas são os menores microrganismos capazes de autoreplicação conhecidos na natureza, responsáveis por uma série de doenças no homem e nos animais, infectando ainda plantas e insetos. Constituem um grande grupo de bactérias, ordenadas em diferentes gêneros na classe Mollicutes, cuja principal característica em comum, além do genoma reduzido, é a ausência de parede celular. Mycoplasma mycoïdes subsp. mycoïdes SC, responsável pela Pleuropneumonia Contagiosa Bovina, foi o primeiro microrganismo desta classe de bactérias a ser identificado. Esta é uma doença bastante grave, com altas taxas de morbidade e mortalidade. A variedade Mycoplasma mycoïdes subsp. mycoïdes LC é responsável principalmente por casos de Pleuropneumonia Contagiosa Caprina, mastite no gado bovino, e ainda artrite em ovinos e caprinos em menor extensão. M. capricolum é um patógeno caprino, responsável principalmente por casos de artrite com grande importância econômica na medicina veterinária. M. pulmonis é um patógeno de roedores, considerado como o melhor modelo experimental para o estudo das micoplasmoses respiratórias. M. genitalium, o menor microrganismo conhecido capaz de se autoreplicar, é um patógeno humano responsável por casos de uretrite não gonocócica, cujo seqüenciamento completo do cromossomo tornou-se um marco na era da genômica. O estudo funcional do genoma destes micoplasmas, para a compreensão de sua biologia e patogenicidade, requer o desenvolvimento de ferramentas genéticas eficientes. No presente trabalho, análises in silico das seqüências na região das prováveis origens de replicação cromossômica (oriC) destes micoplasmas demonstraram a existência de possíveis DnaA boxes localizados em torno do gene dnaA. Estas regiões oriC foram caracterizadas funcionalmente após sua clonagem em vetores artificiais e a transformação dos micoplasmas com os plasmídeos recombinantes resultantes. O plasmídeo pMPO1, contendo a região oriC de M. pulmonis, sofreu integração no cromossomo do micoplasma por recombinação homóloga após poucas passagens in vitro. A redução desta oriC para o fragmento contendo somente os DnaA boxes localizados nas estremidades 5´ou 3´do gene dnaA não foi capaz de produzir plasmídeos replicativos em M. pulmonis, exceto quando estes dois fragmentos foram clonados no mesmo vetor, espaçados pelo determinante de resistência à tetraciclina tetM. Um fragmento interno do gene da hemolisina A (hlyA) de M. pulmonis foi clonado nestes plasmídeos oriC, e os vetores resultantes foram utilizados para transformar o micoplasma. A integração destes vetores por um crossing-over com o gene hlyA, causando a sua dirupção, foi documentada. Deste modo, estes plasmídeos oriC podem vir a se tornar ferramentas genéticas valiosas para o estudo do papel de genes específicos, notadamente aqueles potencialmente envolvidos na patogênese. / Mycoplasmas are the smallest microorganisms capable of self replication known to date, responsible for many diseases in man and animals, infecting also plants and insects. They constitute a large group of bacteria, classified in different genera in the class Mollicutes, which main common characteristic, besides the small genome, is the absence of a cell wall. Mycoplasma mycoïdes subsp. mycoïdes SC, responsible for the Bovine Contagious Pleuropneumonia, was the first microorganism of this class of bacteria to be identified. That is a quite severe disease, with high morbidity and mortality rates. Mycoplasma mycoïdes subsp. mycoïdes LC is responsible mainly for cases of Caprine Contagious Pleuropneumonia, mastitis in cattle, and also arthritis in goats and sheep in less extension. M. capricolum is a pathogen of goats, responsible mainly by cases of arthritis with large economic impact in veterinary medicine. M. pulmonis is a rodent pathogen, considered to be the best experimental model for studying respiratory mycoplasmoses. M. genitalium, the smallest microorganism capable of self replication, is an human pathogen responsible for cases of non gonococcal urethritis, which complete chromosome sequencing has become a benchmark in the era of genomics. Functional studies of these mycoplasma genomes, for comprehension of their biology and pathogenicity, requires the development of efficient genetic tools. In the present work, in silico analysis of sequences of the putative origin of chromosome replication (oriC) region of these mycoplasmas demonstrates the existence of putative DnaA boxes located around the dnaA gene. These oriC regions were functionally characterized after cloning into artificial vectors and transformation of mycoplasmas with the resulting recombinant plasmids. The plasmid pMPO1, which contains the M. pulmonis oriC region, has integrated into the mycoplasma chromosome by homologous recombination after a few in vitro passages. Reduction of this oriC to the fragment containing only the DnaA boxes located upstream or downstream the dnaA gene could not produce plasmids able to replicate in M. pulmonis, except when these two fragments were cloned in the same vector, spaced by tetracycline resistance gene tetM. An internal fragment of the M. pulmonis hemolysine A gene (hlyA) was cloned into these oriC plasmids, and the resulting vectors were used to transform the mycoplasma. Integration of these disruption vectors by one crossing-over with the hlyA gene could be documented. Therefore, these oriC plasmids may become valuable genetic tools for studying the role of specific genes of mycoplasmas, specially those potentially involved in pathogenesis.

L'hétérochromatine, de nature compacte et répétitive, limite l’accès à l'ADN et fait de la réparation des DSBs un processus difficile que les cellules doivent surmonter afin de maintenir leur intégrité génomique. Pour y étudier la réparation des DSBs, nous avons conçu un système CRISPR / Cas9 dans lequel les DSB peuvent être efficacement et spécifiquement induites dans l'hétérochromatine de fibroblastes de souris NIH3T3. En développant un système CRISPR / Cas9 hautement spécifique et robuste pour cibler l'hétérochromatine péricentrique, nous avons montré que les DSB en G1 sont positionnellement stables et réparés par NHEJ. En S / G2, ils se déplacent vers la périphérie de ce domaine pour être réparés par HR. Ce processus de relocalisation dépend de la résection et de l'exclusion de RAD51 du domaine central de l'hétérochromatine. Si ces cassures ne se relocalisent pas, elles sont réparées dans le cœur du domaine de l'hétérochromatine par NHEJ ou SSA. D'autre part, les DSBs dans l'hétérochromatine centromérique activent NHEJ et HR tout au long du cycle cellulaire. Nos résultats révèlent le choix de la voie de réparation différentielle entre l'hétérochromatine centromérique et péricentrique, ce qui régule également la position des DSBs. / Heterochromatin is the tightly packed form of repetitive DNA, essential for cell viability. Its highly compacted and repetitive nature renders DSB repair a challenging process that cells need to overcome in order to maintain their genome integrity. Developing a highly specific and robust CRISPR/Cas9 system to target pericentric heterochromatin, we showed that DSBs in G1 are positionally stable and repaired by NHEJ. In S/G2, they relocate to the periphery of this domain to be repaired by HR. This relocation process is dependent of resection and RAD51 exclusion from the core domain of heterochromatin. If these breaks fail to relocate, they are repaired within heterochromatin by NHEJ or SSA. On the other hand, DSBs in centromeric heterochromatin activate both NHEJ and HR throughout the cell cycle. Our results reveal the differential repair pathway choice between centromeric and pericentric heterochromatin that also regulates the DSB position.