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Stimulation et contrôle de la recombinaison homologue chez le maïs pour augmenter l'efficacité du ciblage de gène et le brassage génétiqueAyar, Ayhan 19 March 2013 (has links)
La recombinaison homologue est un mécanisme de réparation de l’ADN extrêmement contrôlé et particulièrement chez les eucaryotes supérieurs. Dans les cellules méiotiques de ces derniers, où les cassures doubles brin de l’ADN sont programmées, les voies de crossing-over de la recombinaison homologue, qui génèrent de nouvelles combinaisons de gènes, sont restreintes. Dans les cellules somatiques, la recombinaison illégitime, qui assure majoritairement la réparation des cassures double brin de l’ADN, limite l’intégration ciblée du transgène par recombinaison homologue. Les entreprises de biotechnologie convoitent de maitriser la recombinaison homologue afin de contrôler d’une part le brassage génomique qui a lieu pendant la méiose, et d’autre part l’intégration du transgène dans le génome. Cette étude a porté sur le développement d’outils afin d’atteindre ces deux objectifs. Afin d’augmenter le brassage du génome, ayant lieu pendant la méiose, une version du promoteur OsDmc1b, active dans les cellules méiotiques, a été caractérisée chez le maïs. Des plantes sur-exprimant le gène ZmSpo11.1, sous contrôle de ce promoteur, ont ainsi été développées afin d’obtenir des lignées potentiellement hyper-recombinantes. Si la surexpression de ZmSpo11.1 permet effectivement d’augmenter le taux de crossing-over, il pourra être utilisé par les sélectionneurs afin d’accélérer l’introgression d’allèles d’intérêt dans des variétés élites. Concernant la mise en place d’une technique de ciblage de gène, deux stratégies, reposant sur l’utilisation de la méganucléase I-SceI, ont été testées. La démarche a nécessité trois éléments : un locus cible contenant le site de coupure I-SceI, une matrice de réparation et la séquence codant I-SceI (ou I-SceI::GR). La première stratégie, consistant à retransformer les lignées présentant le locus cible avec la matrice de réparation et I-SceI, ne semble pas exploitable car aucun évènement de ciblage de gène n’a été mis en évidence. La seconde stratégie, reposant sur l’assemblage des trois éléments par croisement, est beaucoup plus prometteuse. Malgré la faible activité d’I-SceI::GR, des évènements de recombinaison homologue ont été observés dans les tissus foliaires de certaines plantes. Du cal embryogène, développé à partir de ces dernières, a permis de régénérer des plantes présentant des évènements de ciblage de gène. Ces travaux ouvrent de nouvelles perspectives dans l’élaboration contrôlée d’OGM. / Homologous recombination is a DNA repair mechanism highly regulated in higher eukaryotes. In their meiotic cells, where DNA double-stranded breaks are programmed, the crossing-over pathway of homologous recombination, which generates new gene combinations, is limited in activity and genomic distribution. In somatic cells, illegitimate recombination, which mainly ensures DNA double-strand repair, limits the targeted integration of transgenes by homologous recombination. Biotechnology companies aim to master homologous recombination to control on the one hand the genomic mixing that occurs during meiosis, and on other hand, the integration of transgenes into the genome. This study focuses on the development of tools to achieve these two objectives.To increase genome mixing occurring during meiosis, a version of the OsDmc1b promoter active in maize meiotic cells was isolated. Then, plants over-expressing the ZmSpo11.1 gene under control of this promoter have been developed to obtain potentially hyper-recombinant lines. If ZmSPO11.1 overexpression increases the crossing over rate, it can be used by breeders to accelerate the introgression of alleles of interest into elite varieties. For the establishment of a gene targeting technique, two strategies based on the use of the I-SceI meganuclease were tested. These approaches involved the use of three elements which are: a target locus containing the cleavage site of I-SceI, a repair template and the sequence encoding I-SceI (or ISceI::GR). The first strategy, consisting of the retransformation of target locus lines with the repair template and I-SceI, does not seem workable because no gene targeting events were isolated. The second strategy, based on the assembly of the three components by crossing, is more promising. Despite the low activity of I-SceI::GR, homologous recombination events were observed in leaf tissues of certain plants. Embryogenic callus, developed from these plants, permitted the regeneration of plants with gene targeting events. This work opens new perspectives in the development of controlled GMO production.
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Cellules souches cancéreuses et résistance thérapeutique du cancer du sein : ciblage des cellules souches cancéreuses mammaires par l'inhibition de la réponse au stress réplicatif / Cancer stem cell and therapeutic resistance in breast cancer : targeting breast cancer stem cell by inhibition of DNA replication responseAzzoni, Violette 14 December 2018 (has links)
Les tumeurs mammaires sont connues pour présenter une grande hétérogénéité intratumorale qui contribue à l’échec thérapeutique et à la progression de la maladie. L’origine de dette hétérogénéité s’explique principalement par l’organisation hiérarchique des tissus tumoraux où plusieurs sous-populations de cellules souches de cancer du sein (bCSC) sont capables de s’auto-renouveler et de maintenir l’architecture oligoclonale de la tumeur. Dans la mesure où les bCSC stimulent la croissance tumorale, résistent aux thérapies conventionnelles et initient le développement des métastases, il est indispensable de développer des thérapies spécifiques ciblant ces cellules. L’élaboration d’une telle stratégie nécessite la compréhension des propriétés moléculaires intrinsèques des bCSC. Pour mieux comprendre leur biologie, nous avons isolé les bCSC de différentes xénogreffes dérivées de tumeurs de patientes et établit leurs profil d’expression génique. Nous avons identifié un programme transcriptionnel pouvant être impliqué dans la réduction du stress réplicatif (SR) des bCSC . Nous avons montré que comparé aux non-bCSC, les bCSC présentent une sur-activation de la recombinaison homologue qui leur permet de réduire leur niveau de SR. Nous avons ensuite montré en réalisant un essai clinique que l’inhibition de cette voie permet de les sensibiliser à des agents génotoxique. Ces travaux identifient le SR comme le talon d’Achille des bCSC et mettent en évidence la recombinaison homologue comme cible potentielle pour sensibiliser les BCSC aux thérapies conventionnelles. / Breast tumors are known to present a major intratumoral heterogeneity that contributes to therapy failure and disease progression. The origin of this cellular heterogeneity is mainly explained by a hierarchical organization of tumor tissues where several subpopulations of self-renewing breast cancer stem cells (bCSCs) sustain the long-term oligoclonal maintenance of the neoplasm. bCSCs drive tumor growth, resist to conventional therapies and initiate metastasis development. Thus, developing bCSC-targeting therapies is becoming a major challenge requiring the understanding of the unique molecular circuitry of bCSC as compared to non-bCSC. To better understand the biology of these cells, we isolated bCSCs from different patient–derived xenografts (PDXs), derived fom breast tumors, and established their gene expression profiles. We identified a bCSC core transcriptional program that may be implicated in the reduction of the replicative stress in CSC: overexpression of genes implicated in dNTP metabolism and homologous recombination (HR). Our results show that HR plays a major role in SR regulation of bCSC and that bCSC are more resistant to RS than non-bCSC, We realized a preclinical assay in PDX and showed that HR inhibition prevent bCSC expansion Cisplatin-induced, suggesting a sensitization of the bCSC to the chemotherapy. Our results identify replication stress as the Achilles’ heel of bCSC and highlights HR as potential targets for anti-bCSC therapy.
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Processing Of DNA Recombination And Replication Intermediates By Mycobacterium Tuberculosis RuvA And RuvB ProteinsKhanduja, Jasbeer Singh 02 1900 (has links) (PDF)
Homologous recombination (HR) is a highly conserved cellular process involved in the
maintenance of chromosomal integrity and generation of genetic diversity. Biochemical and genetic studies have suggested that HR is crucial for repair of damaged DNA arising from various endogenous or exogenous assaults on the genome of any organism. Further, HR is vital to repair fatal DNA damage during DNA replication. An instructive example of cross-talk between the processes of DNA recombination and replication can be construed in the processing of replication/recombination/repair intermediates. The impediment(s) to the progression of DNA replication fork is one of the underlying causes for increased genome instability and consequently this might compromise the survival of organism. Various processes manifest at stalled replication forks before they can be rendered competent for the replication-restart. One of the mechanisms of replication-restart involves replication fork reversal (RFR), which envisage unwinding of the blocked forks with simultaneous annealing of the parental and daughter strands o generate a Holliday junction intermediate adjacent to DNA double strand end. Genetic evidence shows that in E. coli dnaEts mutant, holD mutant and in helicase defective rep mutant, RFR is catalyzed by RuvAB complex. Classically, HJ intermediates are generated during the terminal stages of the HR pathway. In E. coli, branch migration and resolution of HJ
intermediates is promoted by RuvA, RuvB and RuvC proteins, which participate at the late stages of HR. Structural, biochemical and mutational analysis suggest that E. coli RuvA binds Holliday junction DNA with high affinity and specificity. RuvB, a member of the AAA+ (ATPase associated with various cellular activities) family, is recruited to the RuvA-Holliday junction complex and functions as a motor protein. Together, RuvA and RuvB catalyze ATP dependent branch migration of HJ. The resolution of HJ is catalyzed by the RuvC endonuclease, which introduces coordinated cuts at two symmetrical sites across the junction.
RuvAB complex, the Holliday junction branch migration apparatus, is ubiquitous in
bacteria. Genetic, biochemical and structural studies have not only established the in vivo role of E. coli RuvAB, in context of HR pathway, but have also provided valuable insights into the mechanism of HJ processing by RuvAB complex. However, the paucity of extensive studies examining the biochemical properties of each member of the RuvABC protein complex restricts models in deciphering the functions of the individual components of this tripartite protein complex. Our current understanding of the biochemical function of E. coli RuvA is within the context of its interacting cellular partner, RuvB. Consequently, the inherent activities of RuvA in the context of DNA repair and HR are poorly understood. Moreover, it remains to be ascertained if RuvABC protein complex, its different sub-complexes, or the individual subunits can function differently in the processing of HJ intermediates generated during DNA repair and HR. The information from these studies would be helpful in understanding the mechanistic details of HR pathway in mycobacteria. Additionally, a number of important questions regarding the molecular basis of RuvAB catalyzed fork reversal remain unanswered. Therefore, exploration of biochemical details of the RuvAB mediated RFR would provide mechanistic insights into the dynamics of fork reversal process. Moreover, analysis of RuvAB catalyzed RFR might be helpful in validating the different assumptions of the RFR model that has been proposed on the basis of genetic analysis of certain E. coli replication mutants. Another interesting question that remains to be answered is, how under in vivo conditions, RuvABC protein complex or its individual subunits are regulated to function differently in the context of HR and DNA repair?
Mycobacterium tuberculosis is an important intracellular pathogen which is likely to
experience substantial DNA damage inside the host and thus may require an efficient DNA recombination and repair machinery for its survival. Our knowledge about the mechanistic aspects of genetic exchange in mycobacteria is rather limited. Therefore, understanding of the processes catalyzed by the components of HR pathway may help in molecular genetic analysis of mycobacteria. Sequence analysis of M. tuberculosis genome, followed by various comparative genomic studies, has revealed the presence of putative homologs of E. coli rec genes but it is not known whether these gene products are able to catalyze the reactions similar to their E. coli counterparts. In M. tuberculosis, the genes encoding for the enzymatic machinery required for branch migration and resolution of HJ intermediates are present. The ruvA, ruvB and ruvC genes form an operon, and are probably translationally coupled. Further, these ruv genes are DNA damage inducible. The transcript level of ruvC is regulated by both RecA dependent and independent mechanisms whereas ruvA and ruvB are induced only through RecA dependent SOS response. During M. tuberculosis infection of host cells, expression of ruvA and ruvB genes is upregulated. We therefore surmise that their gene product might be required for DNA replication, recombination or repair, and would be physiologically relevant under in vivo conditions. However, the details of reactions involved in the processing of HR intermediates and rescue of stalled replication forks in M. tuberculosis remains unknown.
In the initial part of this study, we have investigated the function of M. tuberculosis RuvA protein using Holliday junctions containing either homologous or heterologous core. In the later part, we have explored the ability of M. tuberculosis RuvA and RuvB proteins to catalyze in vitro replication fork reversal.
M. tuberculosis ruvA gene was isolated by PCR amplification and cloned in an expression vector to generate the pMTRA construct. Genetic complementation assays, using the pMTRA construct transformed into E. coli ΔruvA mutant, indicated that M. tuberculosis ruvA is functional in E. coli and suggested that it can substitute for E. coli RuvA in conferring resistance to MMS and survival following UV irradiation. Having established the functionality of M.tuberculosis ruvA, a method was developed for heterologous over-expression and purification of M. tuberculosis RuvA protein (MtRuvA). MtRuvA was purified to homogeneity and the identity of purified protein was verified using western blot analysis using the anti-MtRuvA antibodies. Purified MtRuvA was free of any contaminating endo- or exo-nuclease activity. Biochemical functions of MtRuvA were defined by performing detailed investigations of DNA-binding and Holliday junction processing activities. Substrate specificity of purified MtRuvA was examined,through DNA binding assays, by using oligonucleotide substrates mimicking differentintermediates involved in the pathway of recombinational DNA repair. Purified M. tuberculosis RuvA exhibited high affinity for HJ substrate but also formed stable complex with replication fork and flap substrate. DNase I footprinting of MtRuvA-homologous Holliday junction complex confirmed that MtRuvA bound at the junction center. The DNase I protection conferred by MtRuvA, on homologous HJ, was two-fold symmetric; the continuous footprint was 10 bp longon one pair of symmetrical arms and 7 bp on the opposite pair of arms. In parallel, DNase footprinting of MtRuvA-heterologous Holliday junction complex generated a footprint that encompassed 16 nucleotide residues on each strand of the Holliday junction. Different crystallographic studies have envisaged an important role for RuvA in base pair rearrangement atthe center of the junction. Also, in crystal structure of tetramer of EcRuvA-HJ complex twobases at the junction center were unpaired. To explore if RuvA binding leads to helical distortionof Holliday junction, MtRuvA-HJ complexes were subjected to chemical probing with KMnO4.In case of heterologous HJ, binding of MtRuvA resulted in appearance of sensitive T residues at the junction crossover. By contrast, binding of MtRuvA to homologous HJ rendered the T residues at the junction center and within the homologous core sensitive to oxidation by KMnO4.Taken together, these observations suggested that binding of MtRuvA distorts two base pairs at the junction crossover in heterologous HJ, whereas in case of homologous HJ base pairs distortion extends into the arms of the junction. These observations with KMnO4 probing were independently validated, in real time, by using sensitive to 2-aminopurine fluorescence spectroscopy measurements of MtRuvA-HJ complexes. To follow structural distortions upon interaction with MtRuvA, HJ variants carrying 2-AP substitution were generated for both homologous and heterologous HJ substrate. In each junction species, the 2-AP residue was uniquely present either at the junction center, adjacent to the center or away from the center. Incase of heterologous HJ, binding of MtRuvA resulted in increase of fluorescence emission of2-AP residues located at the junction crossover but not those of 2-AP residues that were present1-2 base pairs away from the junction center. Binding of MtRuvA to homologous HJ resulted in increase of fluorescence emission of 2-AP residues located at the junction crossover. Further, increase in fluorescence emission was also observed for 2-AP residues present within the homologous core or adjacent to the homologous core in a pair of symmetrically related arms. Thus, 2-AP fluorescence results suggested that binding of MtRuvA to homologous HJ causes base pair distortion within and adjacent to the homologous core whereas in case of heterologous HJ the base pair distortion is restricted to the junction center. Together, these results suggest thatMtRuvA causes two distinct types of base pair distortions between homologous and heterologous HJ substrates. To explore the relationship between binding of MtRuvA and alterations in global structure of the junction DNA, we employed the established technique of comparative gel electrophoresis. Analysis of data from comparative gel electrophoresis revealed that MtRuvA, upon binding to the Holliday junctions, converts the stacked-X structure of HJ to square-planar form and stabilizes the same for loading of RuvB rings and subsequent branch migration by RuvAB complex. Our results underline the possible existence of distinct pathways for RuvA function, which presumably depend on the structure and the nature of the DNA repair or HR intermediates. In summary, our results show that binding of MtRuvA to the HJ induced changes in the local conformation of junction, which might augment RuvB catalyzed branch migration. An unexpected finding is the observation that MtRuvA causes two distinct types of structural distortions, depending on whether the Holliday junction contains homologous or heterologous core. These observations support models wherein RuvA facilitates, in a manner independent of RuvB, base pair rearrangements at the crossover point of both homologous and heterologous Holliday junctions.
Although the genetic basis of ruvA ruvB catalyzed RFR in E. coli has been understood in some detail but less is known about the genetic and molecular mechanism of fork reversal in mycobacteria or other organisms. Specifically, to examine if the E. coli paradigm can be generalized to other RuvAB orthologs, we explored the RFR activity of M. tuberculosis RuvAB using a series of oligonucleotides and plasmid-based substrates that mimic stalled replication fork intermediates. This approach might be useful in genetic analysis of factors involved in processing of stalled forks in M. tuberculosis wherein technical difficulties associated with the isolation and characterization of appropriate mutants have limited our understanding of DNA metabolism. Importantly, we have asked the questions as to how the structure at fork junction, extent of reversal and presence of sequence heterology might determine the outcome of RuvAB mediated RFR. The results from this study will be helpful in consolidating the proposed in vivo role for RuvAB complex in fork reversal.
The open reading frame corresponding to M. tuberculosis ruvB gene was PCR amplified
and cloned in an expression vector to generate the pMTRB construct. Genetic complementation assays were performed to assess the functionality of M. tuberculosis ruvB in E. coli ΔruvB mutant. The data from these assays suggested that M. tuberculosis ruvB is active in E. coli and it is able to make functional contacts with E. coli RuvA. Moreover, the efficient alleviation of MMS toxicity in E. coli ΔruvB mutant suggested that M. tuberculosis ruvB might have a role in relieving replication stress generated under specific in vivo conditions. For biochemical analysis, M. tuberculosis RuvB protein (MtRuvB) was over-expressed in a heterologous system and purified to homogeneity. The identity of purified MtRuvB was verified using western blot analysis using the anti-MtRuvB antibodies. Purified MtRuvB was free of any contaminating endo- or exo- nuclease activity. The DNA-binding properties of MtRuvB were analyzed, in conjunction with its cognate RuvA, by using different substrates that are most likely to occur as intermediates during the processes of DNA replication and/or recombination.
MtRuvAB bound HJ, three-way junction and heterologous replication fork with high
affinity but with relatively weaker affinity to flap and flayed duplex substrates. MtRuvB
displayed very weak affinity for linear duplex and failed to bind linear single-stranded DNA. The high affinity of MtRuvB for HJ substrate, in presence of its cognate RuvA, is indicative of direct and functional interaction between RuvA and RuvB. To further test this idea, the catalytic activity of MtRuvB was assayed in the in vitro HJ branch migration assay. In this assay,MtRuvB, in association with its cognate RuvA, promoted efficient branch migration of homologous HJ over heterologous HJ. To decipher the role of MtRuvAB in processing of stalled replication fork we performed in vitro replication fork reversal (RFR) assay using both oligonucleotide and plasmid based model replication fork substrates. Initially, binding of MtRuvAB to different homologous fork (HomFork) substrates was analyzed using the electrophoretic mobility shift assays. MtRuvAB exhibited similar binding affinity towards different HomFork substrates bearing different spatial orientation of nascent leading and lagging strands. To gain insight into the role of MtRuvAB in processing of replication forks, in vitro RFR reactions were carried out using an array of synthetic homologous fork substrates. In all these reactions, MtRuvAB catalyzed efficient fork reversal leading to generation of both parental duplex and daughter duplex. In the kinetics of fork reversal reaction, for all the fork substrates,the accumulation of daughter duplex increased with time whereas the increase in parental or nascent strand DNA was negligible. Taken together, our results suggest that MtRuvAB can efficiently catalyze in vitro replication fork reversal reaction to generate a Holliday junction intermediate thus implicating that RuvAB mediated fork reversal involves concerted unwinding and annealing of nascent leading and lagging strands. Equally important, we demonstrate the reversal of forks carrying hemi-replicated DNA, thus indicating that MtRuvAB mediated fork reversal is independent of symmetry at the fork junction. For understanding the role of RuvAB mediated processing of stalled forks at chromosome level, the fork reversal assays were performed using plasmid derived model “RF” substrate. Fork reversal was monitored by restriction enzyme digestion mediated release of 5’ end labeled fragments of specific size from the fourth arm extruded at the branch point of fork junction. In these reactions MtRuvAB complex was proficient at generating the reversed arm de novo from the RF substrate. Further, MtRuvAB complex catalyzed extensive fork reversal as analyzed by release of linear duplex of2.9 kb from a JM substrate. Use of non hydrolysable analogs of ATP and analysis of restriction digestion mediated release of duplex fragments from the reversed arm suggested that MtRuvAB catalyzed RFR reaction is ATP hydrolysis dependent progressive and processive reaction. MtRuvAB complex catalyzed fork reversal on plasmid substrate that had been linearized thus indicating that MtRuvAB mediated RFR is uncoupled from DNA supercoils in the substrate. Notably, MtRuvAB promoted reversal of forks in a substrate containing short stretch of heterologous sequences, indicating that sequence heterology failed to impede fork reversal activity of MtRuvAB complex. These results are discussed in the context of recognition and processing of varied types of replication fork structures by RuvAB enzyme complex.
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Implication des protéines RECA dans le maintien de la stabilité du génome des chloroplastes d’Arabidopsis thaliana.Vincent, Thierry 06 1900 (has links)
La stabilité génomique des organelles de plantes suscite un grand intérêt dans le domaine de la biologie végétale. En effet, plusieurs études récentes suggèrent que ce type d’instabilité génomique pourrait mener à l’isolation de traits intéressants en l’agronomie. Plusieurs protéines sont d’ailleurs déjà été identifiés comme étant impliqués dans le maintien de la stabilité de ces génomes, tels que MSH1, la famille des POLI, OSB1, les protéines Whirly et les Recombinases A (RECA). Le génome nucléaire d’Arabidopsis thaliana encode trois protéines s’apparentant à la Recombinase A bactérienne et qui sont ciblées à la mitochondrie et/ou au chloroplaste, soit RECA1, RECA2 et RECA3. Globalement, ces gènes partagent une similarité de séquence de 61% avec leur homologue bactérien chez Escherichia coli. Chez les bactéries ces protéines jouent un rôle essentiel dans la recombinaison homologue et sont impliquées dans la réparation de l’ADN. Chez Arabidopsis, il a été démontré que RECA2 et RECA3 sont nécessaires au maintien de l’intégrité du génome mitochondriale. Toutefois leur contribution à la stabilité du génome chloroplastique ainsi que le rôle de RECA1 restent obscures. Le but de ce projet est donc de déterminer la contribution éventuelle des protéines RECA d’Arabidopsis dans la réparation de l’ADN chloroplastique et plus précisément le rôle du gène RECA1. Nous énonçons l’hypothèse que les RECA de plantes se comportent effectivement comme leurs orthologues bactériens en étant impliqués dans la recombinaison homologue.
Dans le cadre de ce projet, nous avons tenté d’isoler des lignées mutantes pour chacun des gènes RECA d’Arabidopsis. En somme, nous avons pu obtenir des lignées convenables pour notre étude que dans le cas du gène RECA1. Ces lignées ont été utilisées pour évaluer la contribution de ce gène à la stabilité du génome du chloroplaste. Ensuite, pour étudier la relation épistatique des gènes RECA1, WHY1 et WHY3, un croisement des différentes lignées mutantes pour ces gènes a été réalisé. Nous avons ensuite étudié la sensibilité de toutes ces lignées mutantes à la ciprofloxacine, un agent causant des bris double brin exclusivement dans les organelles de plantes. Finalement,
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nous avons testé la présence de réarrangements dans le génome du chloroplaste en condition normal ou en présence de stress génotoxique. Nos résultats démontrent que les protéines Whirly et RECA1 sont impliquées dans deux voies de réparation de l’ADN différentes et que les Whirly sont suffisantes pour s’occuper des bris d’ADN double brin en l’absence de RECA1. Nous démontrons également que l’absence de Whirly et RECA1 entraine une forte augmentation de la quantité de réarrangements dans le génome du chloroplaste. De plus nous proposons que la polymérase POLIB est impliquée dans la même voie de réparation que RECA1. Finalement nous proposons un modèle pour expliquer nos résultats et impliquons RECA1 dans un mécanisme de réparation d’ADN et aussi un rôle potentiel dans la réplication. / The stability of plant organelles genomes elicits a great interest in the domain of plant biology. In fact, numerous studies suggest that genomic instability can lead to the isolation of interesting traits in the field of agronomy. Some factors such as MSH1, the POLI family, OSB1, the Whirly proteins and the Recombinase A (RECA), have already been identified has being implicated in the maintenance of genome stability. The nuclear genome of Arabidopsis thaliana encodes three proteins, RECA1, RECA2 and RECA3, that shares a high resemblance with bacterial Recombinase A. They are targeted to the mitochondria and/or to the chloroplast. Globally, these genes share a similarity of sequence of 61% with their bacterial homologue in Escherichia coli. In bacteria these proteins play an essential part in homologous recombination and are implicated in DNA repair. In Arabidopsis, RECA2 and RECA3 have been shown as being essential to maintain the integrity of the mitochondrial genome but their contribution to the stability of the chloroplast as well as the role of RECA1 remains obscure. The goal of this project is to establish the eventual contribution of the Arabidopsis RECA proteins in the repair of chloroplast DNA and more precisely the role of the RECA1 gene. We propose the hypothesis that plants RECA act in the same fashion as their bacterial orthologues by being implicated in homologous recombination.
Within the framework of this project, we have attempted to isolate mutant lines for each RECA gene of Arabidopsis. In the end, we were able to obtain appropriate lines for our study only for the RECA1 gene. These lines were then used to evaluate the contribution of the gene to chloroplast genome stability. Afterwards, in order to study the epistatic relationship between the RECA1, WHY1 and WHY3 genes, a cross between different mutant lines of these genes was realised. We then studied the sensitivity of all of those mutant lines to ciprofloxacine, an agent causing double stranded breaks exclusively in plant organelles. Finally, we evaluated the presence of rearrangements in the chloroplast genome under normal conditions and under the presence of a genotoxic stress. Our
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results show that the Whirly and RECA1 proteins are implicated in two separate pathways of DNA reparation and that the Whirly proteins are sufficient to take in charge DNA double strand breaks generated by the absence of RECA1. We also demonstrate that the absence of Whirly and RECA1 causes an increasein the quantity of rearrangements in the chloroplast genome. Furthermore, we propose that the polymerase POLIB is implicated in the same repair pathway as RECA1. Finally we propose a model to explain our results and implicate RECA1 in a DNA repair mechanism and propose a role for RECA1 in DNA replication.
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Chromatin assembly by CAF-1 during homologous recombination : a novel step of regulationPietrobon, Violena 14 December 2012 (has links) (PDF)
The replication of chromosomes can be challenged by endogenous and environmental factors, interfering with the progression of replication forks. Therefore, cells have to coordinate DNA synthesis with mechanisms ensuring the stability and the recovery of halted forks. Homologous recombination (HR) is a universal mechanism that supports DNA repair and the robustness of DNA replication. Nonetheless, mechanisms regulating HR pathways, such as ectopic versus allelic recombination, remain poorly understood. Another essential pathway for genome stability is the wrapping of newly replicated DNA around nucleosomes, leading to the constitution of a chromatin fibre, which allows the structural organization of the genetic material. In Saccharomyces cerevisiae, deficiencies in chromatin assembly pathways lead to replication forks instability and consequent increase in the rate of HR. Histone chaperones play a crucial role during chromatin assembly, thus I decided to focus on the H3-H4 histone chaperone Chromatin Assembly Factor 1 (CAF-1), to study its role in HR processes in Schizosaccharomyces pombe. Indeed, HR includes a DNA synthesis step and little is known about the associated chromatin assembly. My data excluded a role for CAF-1 in allelic recombination and in the maintenance of forks stability. However, CAF-1 was found to play an important role during ectopic recombination, in promoting chromosomal rearrangements induced by halted replication forks. My data support a model according to which CAF-1 allows the stabilization of early recombination intermediates (D-loop), via nucleosome deposition during the elongation of these intermediates. Doing so, CAF-1 counteracts the dissociation of early recombination intermediates by the helicase Rqh1. Therefore, CAF-1 appears to be part of an equilibrium that regulates stability/dissociation of early steps of recombination events. Importantly, I found that the role of CAF-1 in this equilibrium is of particular importance during non-allelic recombination, revealing a novel regulation level of HR mechanisms and outcomes by chromatin assembly.
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Implication des protéines RECA dans le maintien de la stabilité du génome des chloroplastes d’Arabidopsis thalianaVincent, Thierry 06 1900 (has links)
No description available.
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Understanding the Mechanism of Homologous Recombination in Mycobacterium Tuberculosis : Exploring RecA as an Antibacterial Target and Characterization of Holliday Junction ResolvasesNautiyal, Astha January 2015 (has links) (PDF)
Homologous recombination (HR) is conserved across all three domains of life and is associated with a number of key biological processes. Over the years, numerous genetic, biochemical and structural studies have uncovered important mechanistic details and established a role for HR in DNA damage repair, control of DNA replication fidelity and suppression of various types of cancer. Much of our current understanding of the mechanistic aspects of HR is gained from the study of Escherichia coli paradigm. E. coli RecA is the founding member of a nearly ubiquitous family of multifunctional proteins and is substantially conserved among eubacterial species. During HR, RecA protein promotes homologous pairing followed by strand exchange reaction leading to heteroduplex formation. In addition to HR, RecA is a central component of SOS response, recombinational DNA repair and rescue of collapsed replications forks. Moreover, recent work has suggested that DNA recombination/repair mechanisms might contribute to genome evolution and consequently to the generation of multidrug-resistant strains of the pathogen.
The disease caused by Mycobacterium tuberculosis, endemic in certain regions of the world, is a leading cause of disability and death. A thorough knowledge of the function and interaction of specific HR proteins/enzymes involved in the maintenance of genome integrity is essential in order to elucidate the impact of genome perturbation effects on M. tuberculosis. Toward this end, modulation of RecA protein activity, a central component of HR, represents a potential novel target for design of new drugs because of its involvement in various processes of DNA metabolism. Additionally, small molecule modulators of RecA activity may offer novel insights into the regulation and its role in cellular physiology and pathology. Traditionally, antibiotics have been used to treat infections caused by bacteria. Despite their importance, the development of new antibiotics against M. tuberculosis has considerably decreased over the past several years due to disappointing results in clinical trials. These failures may be due the fact that they suffer from low potency or low cell permeability. Therefore, one of the aims of studies described in this thesis was to test the effect of suramin, a known inhibitor of E. coli RecA, on various biochemical activities of mycobacterial RecA proteins and determine its mechanism of action. Furthermore, the most crucial step in the HR pathway and rescue of collapsed DNA replication forks is the resolution of Holliday junctions and other branched intermediates. Because Holliday junction resolvases are essential for the resolution of different types of DNA recombination/repair intermediates, therefore, we considered it worthwhile to study the genomic expression and biochemical properties of HJRs in M. tuberculosis.
Suramin is a commonly used antitrypanosomal and antifiliarial drug, and a novel experimental agent for the treatment of several cancers. A forward chemical screen assay identified several small molecule inhibitors of E. coli RecA. In this screen, suramin (also called germanin), a polysulfonated naphthylurea, and suramin-like agents were found to inhibit EcRecA catalyzed ATPase and DNA strand exchange activity. However, the mechanism underlying such inhibitory action of suramin and whether it can exert antibacterial activity under in vivo conditions remains largely unknown. In an attempt to delineate the range of suramin action, we reasoned that it might be useful to test its effect on mycobacterium RecA proteins. We found that suramin is a potent inhibitor of all known biochemical activities of mycobacterial RecA proteins with IC50 values in the low μM range. The mechanism of action involves, in part, its ability to disassemble the nucleoprotein filaments of RecA-ssDNA. To validate the above results and to obtain quantitative data, a pull-down assay was developed to assess the effect of suramin on RecA–ssDNA filaments. The data indicated that suramin was able to dissociate >80% of RecA bound to ssDNA. Altogether, these results indicated the effectiveness of suramin in the disassembly of RecA nucleoprotein filament. Next, we sought to test whether suramin binds to RecA by using a CD spectropolarimeter. Significant spectral changes were observed upon addition of increasing concentrations of suramin, indicating alterations in the secondary structure of RecA protein. Additional evidence revealed that suramin impaired RecA catalyzed proteolytic cleavage of LexA repressor and blocked ciprofloxacin-inducible recA gene expression and SOS response. More importantly, suramin potentiated the cidal action of ciprofloxacin and reduced the growth of Mycobacterium smegmatis recA+ strain but not its isogenic recA∆ mutant, consistent with the idea that it acts directly on RecA protein. This approach, which appears as an appealing concept, opens up new possibilities to chemically disrupt the pathways controlled by RecA and treat drug-sensitive as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies.
The annotated genome sequence of M. tuberculosis revealed the presence of putative homologues of E. coli DNA recombination/repair genes. However, it is unknown whether these putative genes have the ability to encode catalytically active proteins or participate in biochemical reactions intrinsic to the process of HR or DNA repair. Studies in the second half of the thesis originated from an in silico analysis for genes that encode functional equivalents of E. coli RuvC HJ resolvase(s) in M. tuberculosis. The central intermediate formed during mitotic and meiotic recombination is a four-way DNA junction, also known as the Holliday junction (HJ), and its efficient resolution is essential for proper segregation of chromosomes. The resolution of HJ is mediated by a group of structure specific endonucleases known as the Holliday junction resolvases (HJR) which have been identified in a wide variety of organisms based on their shared biochemical characteristics. Bioinformatics analyses of the evolutionary relationships among HJ resolvases suggests that HJR function has arisen independently from four distinct structural folds, namely RNase H, endonuclease VII-colicin E, endonuclease and RusA. Furthermore, similar analyses of HJRs identified another family within the RNaseH fold, along with previously characterized RuvC family of junction resolvases. This new family of putative HJRs is typified by E. coli Yqgf protein. The yqgf gene is highly conserved among bacterial genomes. Nuclear magnetic resonance structural studies have disclosed notable structural similarities between E. coli RuvC and YqgF proteins. Utilizing homology-based molecular modelling, YqgF is predicted to function as a nuclease in various aspects of nucleic acid metabolism. Sequence analysis of M. tuberculosis genome has revealed the presence of two putative HJ resolvases, ruvC (Rv2594c) and ruvX (Rv2554c, yqgF homolog). Previous studies have demonstrated that M. tuberculosis ruvC is induced following DNA damage and ruvX is expressed during active growth phase of M. tuberculosis. More importantly, the absence of ruvC increased the potency of moxifloxacin in M. smegmatis. Although, these results imply that the ruv genes play crucial roles in DNA recombination and repair in M. tuberculosis, the biochemical properties of their gene products have not been characterized. In this study, we have isolated M. tuberculosis ruvC and yqgF genes and purified their encoded proteins, M. tuberculosis RuvC (MtRuvC) and M. tuberculosis RuvX (MtRuvX), respectively, to near homogeneity. Protein-DNA interaction assays conducted with purified MtRuvC and MtRuvX revealed that both can bind HJ, albeit with different affinities. However, in contrast to MtRuvC, MtRuvX showed robust HJ resolvase activity. The endonuclease activity of MtRuvX was completely dependent on Mg2+and Mn2+ partially substituted for Mg2+.
Additional experiments showed that RuvX exhibits >2-fold higher binding affinity for HJ over other recombination/ replication intermediates. As demonstrated for other HJRs, MtRuvX failed to cleave static HJ and linear duplex DNA. The cleavage sites were mapped within the homologous core of a branch-migratable HJ. To identify catalytic residues in RuvX, we conducted mutational analysis of an acidic amino acid residue guided by the bioinformatics data. The product of MtRuvXD28N retained full HJ-binding activity, but showed extremely reduced HJ-specific endonuclease activity. Further biochemical characterization revealed that MtRuvX exists as a homodimer in solution. Notably, we found that disulfide-bond mediated intermolecular homodimerization is crucial for the ability of MtRuvX to cleave Holliday junctions, implicating that stable junction binding is necessary to promote branch migration and to create cleavable sites. Analysis of qPCR data suggested that the pattern of yqgF gene expression was similar to those of ruvC and recA genes following DNA damage. Together, these data indicate that ruvX expression is induced by DNA-damaging agents and that RuvX might be functionally involved in recombinational DNA repair in M. tuberculosis.
These findings are all consistent with the idea that RuvX might be the bona fide HJ resolvase in M. tuberculosis analogous to that of E. coli RuvC. More importantly, we provide the first detailed characterization of RuvX and present important insights into the mechanism of HJ resolution, which could be directly linked to the regulation of different DNA metabolic processes, including HR, DNA replication and DNA repair. Overall, this study opens a new avenue in the understanding of HR in this human pathogen, together with elucidation of the function of some of the uncharacterized genes may represent a novel set of recombination enzymes.
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Plasticité des cellules cancéreuses coliques : impact du facteur d'identité tissulaire Cdx2 / Plasticity of colorectal cancer cells : impact of Cdx2, a critical factor for intestinal identityHinkel, Isabelle 11 September 2012 (has links)
Le facteur de transcription homéotique Cdx2 est un suppresseur de tumeurs et son expression est réduite de manière hétérogène dans les tumeurs coliques. Or, le cancer colorectal demeure un problème de santé publique par sa fréquence et sa gravité. Au travers de 3 sous-projets, cette thèse visait à comprendre le mode d’action de Cdx2. Premièrement, la cadhérine Mucdhl a été identifiée et caractérisée en tant que nouvelle cible de Cdx2 : Mucdhl inhibe la croissance des cellules cancéreuses coliques et s’oppose à l’activité de la -caténine. Deuxièmement, un effet inattendu de Cdx2 sur le cytosquelette et la rigidité cellulaire a été montré. Ceci pourrait expliquer comment Cdx2 intervient dans l’organisation structurale des entérocytes ou la migration. En parallèle, une lignée cellulaire rapportrice de l’expression de Cdx2 a été créé qui, après validation, sera un outil précieux pour l’étude des mécanismes moléculaires qui conditionnent l’hétérogénéité tumorale. Par la mise en évidence de nouvelles fonctions et cibles de Cdx2, cette thèse permet de mieux appréhender son rôle physiologique et son action de suppresseur de tumeurs dans l’intestin. / Colorectal cancer is a major public health problem because of its frequency and propensity to metastasize. Cdx2 plays the role of a tumor suppressor and its expression is frequently but heterogeneously reduced in colon tumors. Through three sub-projects, this thesis aimed to better understand the mode of action of Cdx2. First, the Cadherin Mucdhl was identified and characterized as a new direct target gene of Cdx2. Mucdhl was also shown to inhibit the growth of colon cancer cells and to oppose -catenin activity. Second, an unexpected effect of Cdx2 on the cell cytoskeleton and rigidity of a cancer cell monolayer was uncovered. This gives new clues about how Cdx2 contributes to the structural organization and differentiation of enterocytes but also nhibits cell migration. In parallel, a reporter cell line for Cdx2 expression was created. After its validation, this cell line will be a precious tool to study the molecular mechanisms underlying tumor heterogeneity in vivo. Altogether, this thesis unraveled new functions and target genes of Cdx2 that permit to better apprehend its physiological function and its action as a tumor suppressor in the intestine.
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Análises da epidemiologia molecular e evolução de pestivírusWeber, Matheus Nunes January 2016 (has links)
O gênero Pestivirus da família Flaviviridae, compreende vírus de genoma RNA fita simples e polaridade positiva que comumente estão associados a infecções em ruminantes e suídeos. Possui quatro espécies reconhecidas: o vírus da diarreia viral bovina tipo 1 (BVDV-1), o BVDV-2, o vírus da doença da fronteira (BDV) e o vírus da peste suína clássica (CSFV). Além disso, possui possíveis espécies atípicas, sendo o vírus ‘HoBi’-like a mais reconhecida na literatura. Com o objetivo de gerar mais informações acerca da epidemiologia molecular de pestivírus no País e dos mecanismos na evolução de vírus do gênero, o presente trabalho será apresentado sob a forma de seis artigos científicos. Os trabalhos visam a caracterização de pestivírus detectados em bovinos e javalis no Rio Grande do Sul, a identificação de cepas resultantes de provável recombinação homóloga, a análise das variantes virais intrahospedeiro (quasispécies) de animais infectados pelo vírus ‘HoBi’-like, e a comparação da multiplicação in vitro de diferentes cepas de pestivírus em cultivos celulares oriundos de diferentes raças bovinas. No primeiro trabalho, amostras de soro bovino de animais do Rio Grande do Sul foram obtidas junto ao serviço veterinário oficial e submetidas a RT-PCR para detecção de pestivírus seguida de sequenciamento e análise filogenética das amostras positivas, onde 57,6% foram classificadas como BVDV-1 e 42,4% como BVDV-2, sendo constada frequência elevada de BVDV-2 quando comparada a outras regiões no mundo. No segundo trabalho, 40 amostras de javalis cativos obtidas de abatedouro foram testadas utilizando metodologia semelhante a anteriormente mencionada, e foi possível a detecção de uma amostra positiva, que foi classificada como BVDV-2, sendo a primeira evidência molecular do BVDV em javalis na literatura. No terceiro artigo científico, foi pesquisado a ocorrência de recombinação homóloga, utilizando metodologia in silico, em sequências de pestivírus presentes em banco de dados públicos, onde foram descritos novos possíveis eventos de recombinação entre BVDV-1a e BVDV-1b, BVDV-2a e BVDV-2b e CSFV-2.2 com outro genogrupo indefinido. No quarto e quinto trabalhos, animais persistentemente infectados com o vírus ‘HoBi’-like foram gerados experimentalmente e, utilizando RT-PCR seguida de clonagem, sequenciamento e análises in silico, a composição e variações intrahospedeiro das quasispécies virais em circulação foram avaliadas, onde foi possível observar que características individuais do hospedeiro são importantes na seleção de variantes virais e que a nuvem de mutantes aumenta com o passar do tempo, não havando quasispécie dominante. Por fim, no sexto artigo científico, a multiplicação de cepas de BVDV-1a e 1b, BVDV-2a e vírus ‘HoBi’-like foram avaliadas in vitro em células de cultivo primário obtidas de gado europeu, zebuíno e raça mista e comparadas utilizando modelos estatísticos, onde foi possível a observação de ausência de diferenças entre as raças (P=0,88), mas presença de diferença entre indivíduos da mesma raça (P˂0,05). Os resultados aqui apresentados somam informações acerca da epidemiologia molecular, biologia e evolução dos pestivírus, sendo importantes para embasar futuras campanhas de controle e erradicação das doenças causadas por pestivírus. / The genus Pestivirus of the family Flaviviridae, comprises single stranded RNA-viruses that are commonly associated with infections in ruminants and swine. It has four recognized species: bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, border disease virus (BDV) and classical swine fever virus (CSFV). It also has putative atypical species where the ‘HoBi’-like virus is the most highlighted in the literature. In order to generate more information about molecular epidemiology of pestiviruses and important mechanisms in the evolution of the virus genus, this work will be presented in six scientific articles form. The works aimed in the characterization of pestivirus detected in cattle and wild boar in Rio Grande do Sul state, identification of strains resulting from putative homologous recombination, analysis of intrahost viral variants (quasispecies) of animals infected with ‘HoBi’-like viruses, and the comparison of in vitro growth of different pestivirus strains in cell cultures derived from different cattle breeds. In the first study, bovine serum samples of cattle from Rio Grande do Sul state were collected by the official veterinary service and submitted to RT-PCR for detection of pestivirus followed by DNA sequencing and phylogenetic analysis of the positive samples, where 57.6% were classified as BVDV-1 and 42.4% as BVDV-2, which revealed high frequency of BVDV- 2 when compared to other regions worldwide. In the second work, 40 captive wild boar lung samples obtained from slaughterhouse were tested using similar methodology described above, and one sample resulted positive and was classified as BVDV-2, which represented the first molecular evidence of BVDV in wild boars. In the third scientific article, the occurrence of homologous recombination was investigated using in silico methods applied to sequences available in public databases, which are described putative new events between BVDV-1a and BVDV-1b, BVDV-2a and 2b and CSFV-2.2 and undetermined genogroup. In the fourth and fifth studies, animals persistently infected with the ‘HoBi’-like viruses were experimentally generated and using RT-PCR followed by cloning, sequencing and in silico analysis, the composition and intrahost variations of viral quasispecies in circulation were evaluated, where it was observed that individual characteristics of the host are important in the selection of viral variants and the mutant clouds increased overtime. Finally, in the sixth scientific article, the growth of BVDV-1a and 1b, and BVDV-2b, ‘HoBi’-like virus strains were evaluated in vitro using primary cell cultures obtained by taurine, indicine and mixed breed cattle using statistical models, it was possible to observe the absence of differences between cattle breeds (P=0.88), but presence of difference between individuals within the breed (P˂0.05). The results presented herein add information about the molecular epidemiology, biology and evolution of pestiviruses, being important to support future control and eradication programs of diseases caused by pestiviruses.
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Estudos sobre vacinologia e evolução do vírus da cinomose caninaBudaszewski, Renata da Fontoura January 2017 (has links)
O vírus da cinomose canina (CDV) é um importante patógeno de cães domésticos e carnívoros selvagens. A infecção pelo CDV é relevante a nível mundial e está associada com alta morbidade e mortalidade. Em diversos países a cinomose é considerada controlada pelo uso de vacinas, no entanto, no Brasil ainda é endêmica, principalmente devido ao grande número de animais não domiciliados. Além disso, surtos em cães e várias espécies de animais silvestres ocorrem com frequência, dizimando populações ameaçadas. As vacinas vivas atenuadas são seguras para cães, mas seu uso não é aconselhado em espécies altamente suscetíveis à infecção pelo CDV. Também os relatos de surtos de cinomose em cães supostamente vacinados levantam a hipótese de que as vacinas disponíveis no mercado podem não ser eficientes frente a algumas cepas de campo. Com o objetivo de gerar dados acerca dos mecanismos de evolução do CDV e desenvolver e testar a eficácia de uma vacina bivalente inativada contra o vírus da raiva (RABV) e CDV a presente tese será apresentada na forma de dois artigos científicos. Ainda, um artigo de revisão sobre os modelos animais utilizados para obtenção de informações sobre o vírus do sarampo utilizando a infecção de CDV em furões e cães foi publicada e será apresentada na presente tese. No primeiro artigo, foi analisada a ocorrência de recombinação homóloga em genomas de CDV e detectou-se oito possíveis vírus recombinantes, incluindo um evento de recombinação entre uma cepa de campo e uma cepa vacinal atenuada, sugerindo que o uso de vacinação com vírus vivo atenuado pode influenciar a evolução do CDV. No segundo trabalho, uma vacina recombinante bivalente inativada baseada em RABV expressando as glicoproteínas do envelope do CDV, hemaglutinina e proteína de fusão, mostrou-se eficiente na proteção contra infecção por CDV em furões quando utilizado um protocolo prime/boost. Finalmente, foi publicada uma revisão de literatura sobre os modelos animais utilizados para obtenção de informações sobre a patogênese do vírus do sarampo utilizando a infecção com o vírus da cinomose. / Canine distemper virus (CDV) is an important pathogen of domestic dogs and wild carnivores. CDV infection is globally relevant and it is associated with high morbidity and mortality. In several countries, distemper is considered controlled by vaccination, however, in Brazil it is still endemic, mainly due to the large number of non-domiciliated animals. In addition, outbreaks in dogs and various species of wild animals occur frequently, decimating threatened populations. Live attenuated vaccines are safe for dogs, but their use is not advised in species that are highly susceptible to CDV infection. Also, reports of canine distemper in supposedly vaccinated dogs raise the hypothesis that commercially available vaccines may not be effective against some wild type strains. In order to investigate the mechanisms of CDV evolution and to develop and assess the efficacy of an inactivated bivalent vaccine against rabies virus and CDV, this thesis will be presented in the form of two scientific papers. Furthermore, a review article on the animal models used to gain information on measles virus using CDV infection in ferrets and dogs has been published and will be presented in this thesis. In the first paper, the occurrence of homologous recombination in CDV genomes was analyzed and eight possible recombinant viruses were detected, including a recombination event between a wild type strain and an attenuated vaccine strain, suggesting that the use vaccines based on attenuated live virus may influence CDV evolution. In the second study, an inactivated bivalent recombinant vaccine based on RABV expressing CDV envelope glycoproteins, hemagglutinin and fusion protein, proved to be effective in protecting against CDV infection in ferrets when using a prime/boost protocol. Finally, a literature review was published on the animal models used to obtain information on the pathogenesis of measles virus using infection with canine distemper virus.
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