In order to contain many millions, or even billions of base pairs within every nucleus of a eukaryotic cell, DNA must be extensively packaged. This is achieved by association of DNA with packaging proteins, resulting in the formation of chromatin, which can lead to various degrees of compaction. The most extreme form of compaction is the highly condensed mitotic chromosome, formation of which is necessary for proper resolution and segregation of the genetic material during cell division. However, the exact nature of the structure of chromatin within the mitotic chromosome and the factors which regulate it remain subjects of debate and continued investigation. The hybrid cell line F1.1 presents a unique tool for the study of mitotic chromosome structure. This mouse cell line has been observed to present a distinct chromatin structure in mitosis assembled over a large region of DNA inserted into one of its chromosomes and originating from the fission yeast Schizosaccharomyces pombe. Direct comparison of the structure of this distinct region of chromatin with that of the adjacent endogenous chromatin could provide insight into the nature of mitotic chromosome structure as well as the properties of the chromatin which are influencing this structure. Microscopy and Hi-C analyses showed that the mitotic chromatin organising or "scaffold" proteins are not altered over the region of S. pombe chromatin, but that the amount of chromatin organised around these proteins is diminished. In accordance with the "radial-loop" model of mitotic chromosome structure, we put forward a model whereby the S. pombe chromatin is organised into smaller chromatin loops around a constant organising scaffold. Examination of the histone post-translational modifications over the region of S. pombe chromatin revealed it to be highly heterochromatic, with high levels of H3K9me3 and associated factors such as HP1α and 5meC, and low levels of activating marks. Generation of further mammalian - S. pombe fusion cell lines recapitulated both the distinct mitotic structure and the heterochromatic profile of the inserted S. pombe chromatin. However, insertion of S. pombe DNA into a mouse cell by transfection rather than fusion resulted in a large region of S. pombe DNA that lacked both a distinct structure and heterochromatin. These results suggest that H3K9me3- mediated heterochromatin may influence the structure of chromatin in mitosis, leading to an organisation into smaller chromatin loops than non-heterochromatic regions.

Submitted by (ana.araujo@ufrpe.br) on 2017-02-17T16:39:46Z No. of bitstreams: 1 Kaliny Veiga Pessoa da Silva.pdf: 1359915 bytes, checksum: de61e5532d3cfedb773fa796979687fb (MD5) / Made available in DSpace on 2017-02-17T16:39:46Z (GMT). No. of bitstreams: 1 Kaliny Veiga Pessoa da Silva.pdf: 1359915 bytes, checksum: de61e5532d3cfedb773fa796979687fb (MD5) Previous issue date: 2011-02-17 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Manihot genus belongs to Euphorbiaceae family, has about 98 species and native to tropical regions of the Americas, with greatest diversity center in Brazil, with 80% of Manihot species, showing a large vegetative polymorphism and a potential source for cassava breeding programs. Cassava (Manihot esculenta Crantz) is the only commercially cultivated species, with the shoots and the tuber roots used for both human food and animal feed. Cassava roots are also used in the manufacture of flour or in the composition of other products. Karyotypic analysis in mitotic or meiotic cells concerning to chromosomal homology, numerical and structural variations, polyploidy and evolution mechanisms of the karyotypes can provide useful information for breeding programs aimed at achieving improved cultivars. In addition, a karyotype study in many cases contributes to the increase cytogenetic markers that while certain aspects related to horticultural assist in the cultivars characterization. Manihot species are considered allotetraploid, with 2n=36 chromosome and x=9 as basic number. Natural interspecific crosses can be found frequently, making in some cases infertile hybrids. Infertility is not easily detected using phenotypic analysis. However, it is believed that these species undergone diploidization process along the evolution, now showing a meiotic behavior of a diploid. This work aimed the mitotic and meiotic analysis in nine species of the genus Manihot in order to confirm the karyotypic stability described at literature data. Three varieties of cassava and eight wild species were analised. The analysis revealed strong mitotic stability among species regarding the number and chromosome morphology, average size of chromosomes of 1.75 and maximum of two pairs of satellites. The meiosis was regular in wild species and irregular in varieties of M. esculenta 'manipeba', showing univalent, bivalent and trivalent at metaphase-anaphase I, showing typical behavior of a triploid and partly irregular meiosis in 'pornunça', producing polyads in microsporogenesis. An additional study was performed with molecular marker ISSR (Inter simple sequence repeat). Polymorphism was observed in 89.7% among the locus of the species, but as expected, there was a great genetic similarity between varieties of M. esculenta cultivated for the wild species. / O gênero Manihot pertence a família Euphorbiaceae, possui cerca de 98 espécies e é nativo das regiões tropicais das Américas, apresentando um grande centro de diversidade genética no Brasil. Cerca de 80% das espécies de Manihot ocorrem no país, exibindo amplo polimorfismo vegetativo e reunindo potencial para utilização em programas de melhoramento genético do gênero. A mandioca (M. esculenta Crantz) é a única espécie comercialmente cultivada, e dela se aproveita tanto a parte aérea como suas raízes reserva para consumo humano e animal, sendo utilizada na fabricação de farinha ou como parte da composição de diversos outros produtos e subprodutos. Análise cariotípica em células mitóticas ou meióticas em relação a homologia cromossômica, variações numéricas e estruturais, poliploidia e mecanismos evolutivos dos cariótipos podem fornecer informações úteis aos programas de melhoramento que visam a obtenção de cultivares melhoradas. Além disso, um estudo cariotípico em muitos casos contribui para o aumento no número de marcadores citológicos que quando relacionados a determinados aspectos horticulturais auxiliam na caracterização de cultivares. As espécies de Manihot são consideradas alotetraplóides, com 2n=36 e um número básico x=9. Cruzamentos interespecíficos naturais podem ocorrer com certa freqüência produzindo híbridos férteis ou não e, que, nos casos de infertilidade, essa característica pode não ser facilmente detectada por análise fenotípica. No entanto, acredita-se que estas espécies sofreram processo de diploidização ao longo da evolução, apresentando hoje um comportamento meiótico típico de diplóide. Este trabalho realizou a análise mitótica e meiótica em nove espécies do gênero Manihot, a fim de confirmar a estabilidade cariotípica descrita na literatura. Para isso, três variedades de mandioca e oito espécies silvestres foram analisadas. O estudo revelou uma forte estabilidade do cariótipo mitótico entre as espécies quanto ao número e morfologia cromossômica, o tamanho médio dos cromossomos de 1,75 e máximo de dois pares de satélites. A meiose foi regular em espécies selvagens e irregular em variedades de 'manipeba‟ M. esculenta, mostrando univalentes, bivalentes e trivalentes na metáfase, anáfase I, mostrando o comportamento típico de uma meiose triplóides e parcialmente irregular em ' pornunça", produzindo políades na microsporogênese. Adicionalmente foi realizado um estudo molecular com marcador ISSR (Simples sequência interna). Foram observados polimorfismos da ordem de 89,7% entre os lócus das espécies estudadas mostrando uma ampla variabilidade genética entre as espécies do gênero que podem ser fontes importantes de genes a serem empregados em programas de melhoramento da espécie cultivada. Entretanto, como esperado, houve uma grande similaridade genética entre as variedades da espécie M. esculenta em relação as espécies silvestres.

Understanding chromosome structure and genome organization of Culicine mosquitoes can potentially contribute to the development of novel approaches to vector control. However, because of highly repetitive nature of the Aedes and Culex genomes, the structure of their polytene chromosomes is damaged by ectopic contacts that make the analysis difficult. Mitotic chromosomes from imaginal discs of 4th instar larvae of Aedes aegypti were tested as a source for the physical genome mapping for this mosquito. Chromosomes in imaginal discs are 10 times more abundant than chromosomes in nervous ganglia, and they do not accumulate chromosomal mutation as cell line chromosomes do. Prometaphase chromosomes in imaginal discs of Ae. aegypti are 4-5 times longer than metaphase chromosomes and can provide higher resolution for physical mapping. Cold temperature (+16°C) was proven to increase the number of the chromosomes. Hypotonic solution treatment of live larvae was proven to elongate chromosomes and improve banding patterns. We differentially stained these mitotic chromosomes with Giemsa and YOYO-1 to revile the banding pattern. We applied fluorescent in situ hybridization (FISH) procedure developed for human chromosomes to Ae. aegypti chromosomes. A strain from Culex pipiens, Cx. quinquefasciatus and their hybrids from the natural population in Virginia was successfully colonized in the laboratory. This strain can be used as a reliable source for cytogenetic studies. / Master of Science in Life Sciences

La condensation mitotique des chromosomes est l'un des mécanismes assurant la transmission fidèle de l'information génétique. Les complexes condensines et leur association à la chromatine sont nécessaires à cette condensation. Cependant, les mécanismes par lesquels ces complexes s'associent aux chromosomes et contribuent à leur condensation sont mal compris. L'objectif de ma thèse était d'identifier et de caractériser des facteurs de condensation encore inconnus collaborant avec le complexe condensine présent chez S. pombe. Par un crible génétique, nous avons recherché des mutants viables lorsque le complexe condensine est complètement fonctionnel mais morts lorsque ce complexe est partiellement défectif. Nous avons ainsi identifié 7 protéines jusqu'alors jamais impliquées dans la condensation mitotique. Parmi ces dernières, nous avons identifié des protéines impliquées dans le remodelage de la chromatine et des facteurs de transcription comme Gcn5, une HAT très conservée, connue pour son rôle de coactivateur de la transcription ; suggérant un lien entre la condensation et la machinerie transcriptionnelle. Gcn5 s'associe à la chromatine au niveau des promoteurs des gènes où elle acétyle principalement H3K9, H3K14 et H3K18. Sa présence au niveau des promoteurs est directement corrélée avec le niveau de transcription des gènes correspondants. Bien que la majorité de la chromatine soit dé-acétylée et que la présence de Gcn5 soit réduite au niveau des chromosomes en mitose, des traces de H3K9 acétylée persistent au niveau de certains promoteurs. Nos résultats suggèrent que cette acétylation persistante pourrait être liée au recrutement du complexe condensine à la chromatine / From yeasts to human, Condensin is essential for mitotic chromosome condensation. However, how Condensin binds to chromatin and, in this context, shapes mitotic chromosome remain poorly understood. Mappings performed from yeasts to mouse have revealed that condensin is enriched near highly expressed genes along chromosome arms, suggesting that as yet identified features associated with transcription take part in condensin binding to chromatin. To identify factors that collaborate with Condensin we performed a synthetically lethal genetic screen in fission yeast. We searched for mutants that are alive when Condensin is fully functional but dead when Condensin is partly defective. We identified 7 proteins never known for their roles in the mitotic condensation, such as some chromatin remodelling and some transcription factors. All these results were consistent with a link between condensation and transcription. Among theses 7 proteins, we found Gcn5, which encodes a conserved HAT, well known for the role it plays as a transcriptional co-activator. Gcn5 binds to gene promoters where it acetylates mainly H3K9, K14 and K18, and its occupancy correlates with transcription rates. Remarkably, although the bulk of chromatin is de-acetylated and Gcn5 reduced from chromatin upon mitosis entry, traces of Gcn5 dependant H3K9 acetylated persist at condensin binding sites. Here, we provide evidence that Gcn5-mediated histone H3 K9 acetylation could assist the binding of Condensin to chromatin