Copy number of a copia-like retrotransposon was compared in self- and cross-fertilizing taxa of the annual plant genus, Amsinckia. It was hypothesized that variation in copy number between populations with contrasting mating systems could be used to interpret the relative importance of factors influencing copy number (purging of element-induced mutations, ectopic exchange, and the level of heterozygosity). Populations of Amsinckia spectabilis var. microcarpa and A. furcata, two outcrossing taxa, and their self-fertilizing relatives, A. spectabilis var. spectabilis and A. vernicosa were studied. Probes for Southern hybridisation were generated for each respective taxa through the amplification of a conserved region of the retrotransposon sequence. There were no observable differences in the numbers or patterns of hybridised bands between related self- and cross-fertilizing taxa. The retrotransposon family studied may have been inactive before divergence of Amsinckia taxa, or the factors influencing copy number and genome location may be expressed in such a way as to yield no observable differences in copy number between species with contrasting mating systems.

In plants, retrotransposon copy numbers have been shown to vary from several hundreds to several million. The factors that regulate retrotransposon copy number are poorly known. A model by FLAVELL et al. (1997) proposing different effects of selection in genomes with low numbers of copia-like retrotransposons, against genomes with higher copy numbers of this retroelement, was tested. Non-synonymous to synonymous substitutions ratio (d N/dS), together with the frequency of occurrence of stop codons within the reverse transcriptase sequence of copia -like retrotransposons, were analyzed in relationship to copy number estimates of this retrotransposons. Evidence of purifying selection was detected in an enzyme directly involved in transposition. Genomes with less than 10,000 copia-like elements showed significantly smaller dN /dS ratios and fewer stop codons compared with genomes containing more than 10,000 copia-like elements. Genome size also appeared to be correlated with dN/dS ratios and frequencies of stop codons. Heterogeneity of copia-like retrotransposons was not related to dN/dS ratios. Copia-like retrotransposons with different copy numbers inside a single genome showed the same dN/d S ratios. The findings were discussed in relation to different possible selective scenarios in plants with low versus high retrotransposon copy number.

Thesis (M.S.)--Duquesne University, 2004. / Title from document title page. Abstract included in electronic submission form. Includes bibliographical references (p. 91-94) and abstract.

Thesis (M.S.)--Rutgers University, 2008. / "Graduate Program in Plant Biology." Includes bibliographical references (p. 44-46).

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed Nov. 20, 2008). PDF text: 116 p. : ill. (some col.) ; 2 Mb. UMI publication number: AAT 3315053. Includes bibliographical references. Also available in microfilm and microfiche formats.

Made available in DSpace on 2014-06-12T18:02:38Z (GMT). No. of bitstreams: 2 arquivo895_1.pdf: 2338866 bytes, checksum: b0579d16e31ce4eb83ec3e3b93a96401 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / O objetivo desse estudo foi caracterizar bactérias causadoras de infecção hospitalar que apresentam resistência aos dois principais grupos de antibióticos que afetam a síntese da parede celular representados pelos glicopeptídeos e ß-lactâmicos. Neste, reportamos o isolamento e caracterização das primeiras amostras de Enterococcus faecalis resistentes ao glicopeptideo vancomicina (VRE) no Nordeste do Brasil e isolados clínicos de Klebsiella pneumoniae resistentes aos antibióticos ß-lactâmicos envolvidos em episódios de infecções hospitalar. Estes isolados foram testados quanto a susceptibilidade aos antimicrobianos de escolha. Os enterococos foram tipificados por meio de macro-restrição do DNA cromossomal e analisados quanto a presença dos genes de resistência e de elementos genéticos móveis. Os isolados apresentaram resistência a múltiplos antimicrobianos e apenas um perfil genético foi encontrado, sugerindo a disseminação de uma linhagem clonal entre os pacientes infectados, também a presença específica do gene vanA e do elemento de transposição Tn1546-like associado a plasmídios mostram capacidade de disseminação dos mecanismos de resistência aos glicopeptídeos por meio de processos de conjugação genética entre as bactérias. Os isolados de K. pneumoniae foram tipificados por ribotipagem e analisados quanto a presença dos genes de resistência e de elementos genéticos móveis. Os isolados apresentaram o fenótipo de ESBL, ou seja, produtoras de ß-lactamases de espectro ampliado. Este fenótipo foi associado a presença dos genes blaTEM, blaCTX-M e blaSHV tanto no cromossomo como em plasmídios naturais dos isolados. Estes genes foram detectados em diferentes associações, como genes únicos (25,5%), duplos (41,8%) e triplos (32,7%). A presença dos três genes no mesmo isolado foi detectada em percentuais superiores àqueles relatados na literatura. Em adição, foram também detectadas as presenças do integron de classe 1 carreando outros genes de resistência, gerando o fenótipo de co-resistência dessas bactérias a outros antimicrobianos. A detecção de várias linhagens clonais mostra o surgimento de vários eventos de resistência ou a transferência dos marcadores genéticos entre as bactérias. Assim, os resultados puderam mostrar o crescimento da disseminação dos elementos genéticos de resistência bacteriana a antibióticos entre isolados de bactérias causadoras de infecção hospitalar, diminuindo, assim, as possibilidades terapêuticas para o tratamento dessas infecções

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Les transposons sont des séquences d’ADN qui ont la capacité de se dupliquer de façon autonome, posant une menace pour l’intégrité et la stabilité du génome. De nombreux mécanismes existent pour contrôler l’expression des transposons, parmi lesquels la méthylation de l’ADN joue un rôle particulièrement important. Chez les mammifères, les profils de méthylation sont stables tout au long de la vie de l’individu, mis-à-part pendant deux moments clés du développement embryonnaire. Pendant ces deux périodes, la méthylation de l’ADN est globalement effacée, ce qui corrèle avec l’acquisition d’un état cellulaire pluripotent, puis rétablie. En utilisant un système cellulaire de reprogrammation de méthylation induite, ce travail s’est attaché à comprendre comment le génome parvient à maintenir le contrôle des transposons en l’absence de cette protection d’ordinaire essentielle, J’ai pu démontrer que divers mécanismes chromatiniens compensent progressivement la disparition de la méthylation de l’ADN pour le maintien de la répression des transposons. En particulier, la machinerie Polycomb prend en partie le relai et acquiert un rôle primordial, spécifiquement en l’absence de méthylation de l’ADN. Dans un second temps, la contribution du cofacteur d’ADN méthyltransférase DNMT3l lors de la méthylation de novo a été étudiée. Dans sa globalité, ces découvertes offrent des perspectives nouvelles sur la façon dont le génome se réorganise lors de moments clés du développement embryonnaire. / Transposons are DNA sequences that can duplicate autonomously in the genome, posing a threat for genome stability and integrity. To prevent their potentially harmful mobilization, eukaryotes have developed numerous mechanisms that control transposon expression, among which DNA methylation plays a particularly important role. In mammals, DNA methylation patterns are stable for life, at the exception of two key moments during embryonic development, gametogenesis and early embryogenesis. After a phase a global loss of genomic methylation accompanying the acquisition of pluripotent states, DNA methylation patterns are re- established de novo during differentiation. This work attempted to elucidate how the genome copes with the rapid loss of DNA methylation, in particular regarding the control of transposons in absence of this essential protective mark. Using an embryonic cellular model of induced methylation reprogramming, I showed that various chromatin-based mechanisms can compensate for the progressive loss of DNA methylation. In particular, my results suggest that the Polycomb machinery acquires a critical role in transposon silencing, providing a mechanistic relay specifically when DNA methylation patterns are erased. In a second phase, this work analyzed the contribution of the DNA methyltransferase cofactor DNMT3l during events of embryonic de novo methylation. Overall, these findings shed light onto the processes by which genome regulation adapts during DNA methylation reprogramming.

Transgenesis is a process of introducing foreign genetic material into the genomes of living organisms. One of the tools for transgenesis are the transposable elements (TEs), which include transposons. Transposons are naturally occurring sequences of DNA which are recognised, excised and inserted into a new location by a single enzyme – transposase. Here we show studies of the biophysical properties and activities of two highly related eukaryotic TEs of the mariner family: Mos1 from Drosophila mauritiana and Mboumar-9 (Mbo9) from Messor bouviery. Using biochemical and molecular methods we examined the properties of transposases in vitro and in vivo. Recombinant transposases were expressed in E.coli and purified using HPLC. Each protein’s activity was assayed for cleavage, integration and the whole transposition reaction. We used a modelling approach to predict the structure of the complex of Mbo9 transposase bound to the specific terminal sequences of the transposon, the paired end complex (PEC), based on the published crystal structure of Mos1 PEC. We have found that both transposases are elongated dimers in solution and that the first helix-turn-helix domain is involved in the protein dimerization. Moreover we show that mariner transposases cut one of the imperfect inverted repeats more efficiently than the other. The terminal nucleotide of the inverted repeat is important for integration of the transposon into a new target DNA, while having no effect at the stage of cleavage. Previously, neither Mos1 nor Mbo9 had been shown to have significant activity in mammalian cells. We have developed a new assay that allows chromosomal integration of the desired DNA sequence in vivo in bacterial, yeast and mammalian cells without the use of helper plasmids or mRNA injection. We found the optimal combination of inverted repeats for each of the transposons and have enhanced the transposition efficiency of Mbo9 by changing the sequence of its inverted repeat DNA. This study is a foundation for improving mariner TEs for transgenesis.