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Stable propagation of the yeast 2 micron plasmid : equal segregation by hitchhiking on chromosomes.Chang, Keng-Ming 24 June 2014 (has links)
The 2 micron plasmid of Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number of 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-encoded partitioning system and an amplification system to ensure its stable, high-copy propagation. The partitioning system consists of the plasmid encoded proteins, Rep1 and Rep2 and a cis-acting partitioning locus STB. The Rep proteins, together with several host factors, assembled at STB couple plasmid segregation to chromosome segregation. A plasmid lacking an active partitioning system is subject to a ‘diffusion barrier’, which causes it to be retained in the mother cell with a strong bias (mother bias). Currently available evidence favors the hitchhiking model for plasmid segregation, in which the tethering of plasmids to chromosome provides the basis for faithful plasmid partitioning. However, direct evidence to support this hypothesis has been difficult to obtain because of the small size of the budding yeast nucleus and the poor resolution of chromosomes in live cells or in chromosome spreads. In this study, we have attempted to verify the hitchhiking model using single copy derivatives of the 2 micron plasmid as reporters. We demonstrate, using two single copy reporters present in the same nucleus, that plasmid association with chromosome spreads is authentic, and is dependent on the partitioning system. By using a strategy that forces all chromosomes to stay in either the mother or the daughter compartment, we show that plasmid segregation can be uncoupled from nuclear envelope segregation. However, plasmid segregation cannot be uncoupled from chromosome segregation under this condition. This tight coupling between plasmid and chromosome segregation is consistent with the hitchhiking model for plasmid segregation. The plasmid partitioning complex is assembled de novo at STB during each cell cycle during the G1-S window. Plasmid replication or cell cycle cues that signal cellular DNA replication appear to trigger this assembly. Furthermore, there is an apparent temporal hierarchy in the association and dissociation of protein factors at STB. When DNA replication is delayed or blocked, the dissociation of factors from STB from the previous portioning cycle and the association of factors for the new partitioning cycle are delayed or blocked, respectively. The precise role of replication in plasmid segregation has not been elucidated. We have addressed this question by blocking either plasmid replication or all cellular DNA replication. We find that replication is not required for plasmid to overcome mother bias. However, replication is critical for the equal segregation of sister plasmid copies. These results provide a refinement of the hitchhiking model by suggesting that sister plasmids tether to sister chromatids in a replication-dependent manner and hitchhike on them during chromosome segregation. Finally, we have attempted to reconstitute the 2 micron plasmid partitioning system in mammalian cells with the goal of exploiting their larger nuclear size and the considerably higher chromosome resolution they provide. In experiments completed so far, we show that Rep2 expressed in COS7 cells localizes to chromosomes, and Rep1 does so in the presence of Rep2. Furthermore, they show co-localization on sister chromatids in a symmetric fashion, implying that plasmids associated with them are likely to follow suit. These observations suggest, by extrapolation, the Rep1-Rep2 assisted association of sister plasmids with sister chromatids in yeast as well, and are consistent with the refined hitchhiking model for plasmid segregation. / text
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The segregation of native and foreign extra-chromosomal genetic elements in Saccharomyces cerevisiae : stable propagation by hitchhiking on chromosomesLiu, Yen-Ting, 1980- 07 November 2013 (has links)
The 2 micron plasmid of the budding yeast Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number around 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-coded partitioning system and an amplification system to exhibit nearly chromosome-like stability in its host. Plasmid behavior under conditions that missegregate chromosomes suggest that the partitioning system couples plasmid segregation to chromosome segregation. However, the mechanism of this coupling has not been elucidated. A plausible model, consistent with current evidence, is the hitchhiking model, in which plasmid-chromosome tethering provides the basis for faithful plasmid partitioning. Testing this hypothesis unequivocally has been difficult, primarily because of the technical limitations posed by the small size of the budding yeast nucleus and poor resolution of chromosomes. As a result, cell biological assays based on fluorescence microscopy have had only modest success in addressing this problem. In the present study, I devised an experimental verification of the hitchhiking model using a single copy derivative of the 2 micron plasmid as a reporter. The rationale was to establish various conditions that force sister chromatids to co-segregate during mitosis in a bias-free manner or with a bias towards the daughter. The segregation patterns of plasmid sisters were followed under these conditions. The sum of the results from this analysis is accommodated by the hitchhiking model, with sister plasmids associating with sister chromatids in a one-to-one fashion. Episomes of mammalian viruses belonging to the gamma-herpes and papilloma families utilize a hitchhiking mechanism to persist in cells during the latent phase of their infection. Two of the viral partitioning systems have been reconstituted in S. cerevisiae. We wished to exploit these systems to characterize the efficiency of non-native chromosome tethering systems in promoting equal segregation of viral plasmids in S. cerevisiae. We find that the 2 micron plasmid partitioning system is considerably superior to the viral systems. This could be due to the higher efficiency of plasmid-chromosome association and/or due to the ability of plasmid sisters to tether to sister chromatids. / text
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Advancing the Safety of Lentiviral Vector Mediated Gene TherapyShaw, Aaron Marcus 04 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Lentiviral vector mediated gene therapy has made great strides in recent years with several successful clinical trials. However, adverse events encountered with some early trials have highlighted the necessity to improve upon its safety. Improvements can range from early steps in vector production to evaluation of insertion sites post-transduction. We have evaluated an FDA approved DNase for removal of residual plasmid DNA during vector production, developed novel non-integrating lentiviral vectors and employed modified insertion site analysis post-transduction to improve the safety of lentiviral vector mediated gene therapy.
To prevent the exposure of gene therapy patients to HIV-1 DNA it is essential to remove residual plasmid DNA during vector production. We evaluated a recombinant human DNase which has been FDA approved for use in patients as an alternative to a bacterially derived DNase. Our results indicate this DNase is an effective alternative with a potentially safer profile for use in patients.
The ability of lentiviral vectors to stably integrate their genome into a host cell’s DNA can have negative side-effects due to the risk of insertional mutagenesis. Non-integrating lentiviral vectors have been developed to alleviate this risk in applications where integration is not necessary. However, a low frequency of illegitimate integration persists when using these vectors. We have developed a novel non-integrating vector mutation and evaluated the efficacy of combining it with other mutations for reducing the frequency of illegitimate integration. We demonstrate that combining mutations that inhibit integration can further reduce the frequency of illegitimate integration.
Several methodologies have been developed for evaluating the insertion sites of normal integrating lentiviral vectors. Illegitimate integration by non-integrating vectors demonstrates mechanisms which result in insertions and/or deletions at the vector-genome junction. Current methods lack the sensitivity to account for these variables in a high-throughput manner. We have adapted modifications to current methods to improve the capture of these variable insertion sites for analysis.
The results of these studies improve the safety of lentiviral vector mediated gene therapy by improving the purity of the vector product, providing a safer vector for non-integrase mediated applications, and allowing more sensitive analysis of insertion sites post-transduction.
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Οχήματα γονιδιακής μεταφοράς για τη γονιδιακή θεραπεία των αιμοσφαιρινοπαθειών / Gene transfer vehicles for the therapy of hemoglobinopathiesΠολυβίου, Σταύρος 29 June 2007 (has links)
Τα επισωματικά οχήματα γονιδιακής μεταφοράς αποτελούν ελκυστική εναλλακτική πειραματική προσέγγιση της γονιδιακής θεραπείας σε σχέση με τα ιϊκά οχήματα. Στην παρούσα εργασία και στο πλαίσιο της ανάπτυξης επισωματικών οχημάτων για την γονιδιακή θεραπεία των αιμοσφαιρινοπαθειών, μελετάται το όχημα hβ-SMAR(Α), ένα κυκλικό πλασμίδιο, που φέρει το γονίδιο της ανθρώπινης β-σφαιρίνης και το μLCR και βασίζεται σε ανθρώπινα χρωμοσωμικά στοιχεία, το S/MAR στοιχείο από την περιοχή 5’ του γονιδίου της ανθρώπινης ιντερφερόνης β. Διαπιστώθηκε ότι το hβ-SMAR(Α) συγκρατήθηκε εντός της διαμολυσμένης κυτταρικής σειράς MEL για περισσότερες από 300 γενιές με διαπιστωμένη την επισωματική του κατάσταση για τουλάχιστο 180 γενιές. Επιπλέον, διατήρησε υψηλά επίπεδα έκφρασης του διαγονιδίου, τα οποία θα αντιστοιχούσαν σε θεραπευτικά επίπεδα, αν αναπαράγονταν in vivo. / Episomal vehicles for gene transfer are an attractive alternative experimental approach to gene therapy in the place of viral vectors. The vehicle hβ-SMAR(Α) studied here, within the context of developing efficient episomal vectors for the gene therapy of hemoglobinopathies, is a circular plasmid bearing the human β globin gene and the μLCR and is based on human chromosomal elements, the S/MAR element from the region 5’ of the human interferon β gene. It was established that hβ-SMAR(Α) was retained within the transfected MEL cell line for more than 300 generations, with its episomal state ascertained for at least 180 generations. Furthermore, it retained high level expression of the transgene, which would be therapeutic, if reproduced in vivo.
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Rôle de l'interaction entre la protéine virale EBNA1 et le facteur cellulaire RCC1 dans la persistance du génome du virus d'Epstein-Barr / Role of the interaction between the viral protein EBNA1 and the cellular factor RCC1 for the persistance of the Epstein-Barr Virus genomeDeschamps, Thibaut 18 September 2015 (has links)
Le virus d’Epstein-Barr (EBV) est un herpesvirus dont la séroprévalence est d’environ 90 % de la population adulte mondiale. EBV est associé à de nombreuses pathologies tumorales. La primo infection conduit à l’établissement du virus sous forme latente dans les lymphocytes B mémoires. Au sein de ces cellules B, le génome viral est sous la forme d’un épisome, un ADN circulaire double brin, et une fraction restreinte de gènes viraux est exprimée. Afin de se maintenir aux cours des divisions cellulaires, le génome viral est répliqué en phase S par la machinerie cellulaire et ségrégé lors de la mitose dans chaque cellules filles. La réplication et la ségrégation du génome viral nécessitent 2 facteurs viraux que sont la protéine virale EBNA1 (Epstein-Barr Nuclear Antigen 1) et la région oriP sur le génome viral. En phase S, EBNA1 interagit directement avec l’oriP et y recrute le complexe de pré-réplication de l’ADN. En mitose, EBNA1 ancre l’épisome à la chromatine ce qui permet une ségrégation efficace. Les mécanismes d’interaction entre EBNA1 et la chromatine reste encore flou. Au cours de notre travail, nous avons identifié la protéine RCC1 comme un partenaire potentiel pour la protéine EBNA1 pouvant être impliqué dans l’ancrage d’EBNA1 à la chromatine. Nous avons validé cette interaction et caractérisé les régions d’interactions pour ces deux protéines. Par ailleurs nous avons démontré que RCC1 est recrutée sur l’oriP en présence d’EBNA1 et que ces deux protéines interagissent en mitose. À la lumière de nos résultats et des données de la littérature, nous proposons que l’interaction d’EBNA1 avec la chromatine est dynamique et implique à la fois des interactions directes (AT-Hook, interaction avec les nucléosomes) mais aussi des facteurs cellulaires (RCC1, EBP2 et HMGB2). / Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with several human cancers. In proliferating latently-infected cells, the EBV genome persists as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both of these processes require a single viral protein, Epstein Barr nuclear antigen 1 (EBNA1), which binds to two clusters of cognate binding sites within the origin of plasmid replication (oriP). EBNA1 plays an essential role both in viral episome replication, by recruiting the cellular complex of DNA replication onto the oriP, and in the efficient segregation of the viral episomes, by tethering the viral DNA onto the mitotic chromosomes. Whereas the mechanisms of viral DNA replication have been well documented, the mechanisms involved in tethering EBNA1 to the cellular chromatin are far from being understood. Here we have identified Regulator of Chromosome Condensation 1 (RCC1) as a novel EBNA1 cellular partner. RCC1 is the only known nuclear guanine nucleotide exchange factor (RanGEF) for the small GTPase Ran enzyme. RCC1, associated with chromatin, is involved in the formation of RanGTP gradients critical for nucleo-cytoplasmic transport, mitotic spindle formation, and nuclear envelope reassembly after mitosis. We have used several approaches to demonstrate a direct interaction between these two proteins and to identify the regions. involved Moreover, by using Chromatin ImmunoPrecipitation assay (ChIP) we have shown that RCC1 is enriched in the oriP region of mini viral replicons in a manner dependent on EBNA1. Finally, by using a combination of confocal microscopy and FRET analysis to follow the dynamics of interaction between the two proteins throughout the cell cycle, we have demonstrated that EBNA1 and RCC1 closely associate on the chromosomes during metaphase. Taken together, our data strongly suggest an essential role for RCC1 in tethering EBNA1 - linked to the viral episome - to the metaphasic chromosomes. Our results and those of others lead us to the idea that the interaction between EBNA1 with the cellular chromosomes requires several factors such as direct interactions or cellular proteins and these interactions are complementary and / or redundant.
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