<p>Heterochromatin, or condensed chromatin, is a transcriptionally repressive form of chromatin that occurs in many eukaryotic organisms. At its natural locations, heterochromatin is thought to play important roles in genome organization as well as gene expression. Just as important is the restriction of this repressive form of chromatin to appropriate regions of the genome. In the budding yeast <italic>Saccaromyces cerevisiae</italic>, domains of condensed, transcriptionally silenced chromatin are found at telomeres and at the silent-mating type cassettes, <italic>HML<italic/> and <italic>HMR</italic>. At these locations, a complex of Silent Information Regulator (SIR) proteins gets recruited to DNA through discrete silencer elements. Once recruited, the Sir protein complex then spreads along chromosomes in a step-wise manner. This process results in the silencing of gene expression. It is unclear whether silenced chromatin is established in the same manner at different genomic locations. Understanding how silenced chromatin is formed is important for determining how these chromatin structures are regulated.</p><p>To better understand how silenced chromatin is established in different genomic contexts, I used chromatin immuoprecipitation to follow the rate of silenced chromatin formation at different locations. The rates of Sir protein assembly were compared at two locations, telomere VI-R and <italic>HMR</italic>. I discovered that the silencers at these two locations were equally proficient at recruiting Sir proteins. However, the rate of Sir protein assembly onto nucleosomes was far more rapid at <italic>HMR</italic> than at the telomere VI-R. Furthermore, the rate of Sir protein assembly was more rapid on one side of the <italic>HMR-E</italic> silencer at <italic>HMR</italic> than the other. Moreover, insertion of the <italic>HMR-E</italic> silencer adjacent to the telomere VI-R significantly improved the rate of Sir protein assembly onto nucleosomes. Additionally, observations that the association of Sir protein occurs simultaneously across several kilobases at <italic>HMR</italic> and that silencing at <italic>HMR</italic> is insensitive to co-expression of wild-type and catalytically inactive Sir2 proteins suggest that <italic>HMR-E</italic> enables the assembly of silenced chromatin in a non-linear fashion. These results suggest that <italic>HMR-E</italic> functions to both recruit Sir proteins and promote their assembly across several kilobases.</p><p>In addition to the <italic>HMR-E</italic> silencer, <italic>HMR</italic> is also characterized by the presence of a second auxiliary <italic>HMR-I</italic> silencer and a tRNA gene that functions as a boundary element to restrict the spread of silenced chromatin. I used chromatin immunoprecipitation to determine how each of these regulatory elements contribute to the steady-state levels of Sir protein association with chromatin. Consistent with a role for <italic>HMR-E</italic> beyond recruitment, I discovered that the <italic>HMR-E</italic> silencer alone promoted higher levels of Sir proteins on nucleosomes compared to the telomere VI-R. The levels of Sir protein association with <italic>HMR</italic> were further elevated by the <italic>HMR-I</italic> silencer, even though this silencer does not recruit Sir proteins on its own and does not contribute to any of the known functions of silenced chromatin at <italic>HMR</italic>. Additionally, although the tRNA gene did block the spread Sir proteins, I discovered that the capacity for Sir proteins to spread beyond a few kilobases was severely limited even in the absence of the boundary.</p><p>The results of this thesis work provide new insights into the mechanisms of silenced chromatin establishment and regulation in budding yeast. I show here that the capacity of Sir proteins to assemble onto nucleosomes is inherently limited. Additionally, silencers vary in their ability to promote this assembly. I conclude that the silencer is a key factor in determining the relative size, efficiency, and location of silenced chromatin domains in the cell.</p> / Dissertation

Chromatin is a multi-layered structure composed of DNA, nucleosomes, histone modifications, and associated proteins that critically affects genome function. Recently developed sequencing technologies enable genomewide characterization of certain aspects of chromatin structure, including nucleosome positioning and histone modifications. However, chromatin proteins present several challenges due to their dynamic nature and variable association with DNA. Chromatin proteins such as Polycomb regulators and heterochromatic factors play critical and global roles in epigenetic repression and hence new approaches are needed for their study. We first sought to identify sequences that recruit Polycomb repressive complex 2 (PRC2) in mammalian cells. We combined chromatin immunoprecipitation with sequencing (ChIP-seq) to map the candidate transcription factor YY1, and found that it does not correlate with PRC2 localization, suggesting that YY1 is not directly involved in PRC2 recruitment. We also identified GC-rich sequences that are necessary and sufficient for PRC2 recruitment. Yet attempts to map additional Polycomb proteins and other repressors using ChIP-seq proved difficult. Since chromatin proteins are often broadly, secondarily or transiently bound to DNA, they are difficult to crosslink. Antibody quality also varies, further hampering ChIP-seq technology. Here, we adapt DamID, a method for mapping chromatin regulators that uses a fusion enzyme and that does not rely on crosslinking or antibodies, for high-throughput sequencing. We show that DamID-seq can be used to globally characterize chromatin repressors in human cells. We used DamID-seq to map the binding of 12 chromodomain-containing and related proteins in K562 cells. We found that these proteins cluster into two modules: 1) Polycombrelated and 2) heterochromatin-related. Polycomb proteins bind developmental genes, while heterochromatin proteins bind broad olfactory receptor (OR) and zinc finger (ZNF) domains. Surprisingly, unlike other Polycomb proteins, CBX2 uniquely binds genes involved with modifying proteins. Our findings advance the model that the genome is compartmentalized into domains, and identify the distinct protein components that associate respectively with Polycomb and heterochromatin domains in human cells. We expect that DamID-seq, along with further advancements in characterizing the three-dimensional organization of chromatin, will bring us towards a better understanding of the role of chromatin in differentiation, development, and disease.

Epistasis describes non-additive interactions which affect gene expression and phenotype. It can happen on multiple levels, including on a genomic level with interactions between genes or even chromosomes affecting global patterns of gene expression. It can also happen within a gene itself, with epistatic interactions between amino acids affecting gene expression and resultant phenotypes. I present three studies in two organisms to study this phenomenon on a global-genomic scale, and also on a local-genic scale.

No description available.

HOAP was originally identified as a component of an ORC-containing multi-protein complex of Heterochromatin Protein 1 (HP1) from early Drosophila embryos. HOAP immunostaining showed prominent association of it with telomeres, and mutants for HOAP (cav1) showed it functions along with HP1 in forming a telomere capping complex that prevents telomeric fusions. Weaker HOAP immunostaining is also observed in regions of pericentric heterochromatin and euchromatin. To examine the role of HOAP at these non-telomeric sites, we applied Affymetric Drosophila Genome Arrays to undertake a microarray expression profiling study of genes that are mis-expressed in cav1 mutant larvae. The data from four publicly available databases were used to assess the normal expression patterns of the affected genes. We found that the majority (67%) of genes with decreased expression levels in cav1 mutants (log2R< -2.0, pvalue≤ 0.01) have normally testis-specific expression. These results could indicate a role of HOAP in testis-specific gene expression. Alternatively they could reflect reduced male viability due to the loss of HOAP, which resulted in the under-representation of males in the cav1 larval sample. The latter hypothesis is supported by the observation of 2.8-fold under-representation of males in cav1 larvae when I used a yellow+-marked X chromosome to differentially mark male and female cav1 larvae. Thus, this project is focused on determining and characterizing the cause of the reduced male viability. Here I report a role for both HOAP and HP1 in regulating the establishment promoter, SxlPe, of the sex determination masterswitch, Sex lethal (Sxl). Female-specific activation of SxlPe is essential to females as it provides SXL protein to initiate productive female-specific splicing of the late Sxl transcripts which are transcribed in both sexes. We find inappropriate firing of SxlPe and splicing of Sxl transcripts in male cav mutants, whereas mutants for HP1 display Sxl splicing defects in both sexes. Both proteins are associated with SxlPe sequences. In embryos from HP1 mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to SxlPe is severely compromised. Our genetic and biochemical assays suggest a repressing activity for HOAP and both activating and repressing roles for HP1 at SxlPe.

In Drosophila there are two different chromosome-wide targeting systems, the dosage compensation system that equalizes the transcriptional output from X-linked genes between males and females, and the regulation of the 4th chromosome mediated by the POF protein.   The best studied of these two mechanisms is the dosage compensation system. To attain dosage compensation in Drosophila at least five different proteins, encoded by the male-specific lethal genes msl1, msl2, msl3, mle and mof, are required. These proteins together with two non-coding RNAs (roX1 and roX2) form a dosage compensation complex (MSL complex), which binds exclusively to the X chromosome in Drosophila males and up-regulates the transcription approximately two times.   In this thesis I show that roX1 and roX2 are most likely the only non-coding RNAs within the MSL complex. As expected, the roX transcripts were enriched within the MSL complex. Interestingly, one additional transcript was identified within the MSL complex. This transcript did not associate with the X chromosome and is therefore not believed to be involved in up-regulation of the X-linked genes. This transcript encodes for the rate limiting component in the MSL complex, the MSL2 protein. A model is proposed in which free, partial or complete, MSL complex feed-back regulates the amount of msl2 transcript, and thereby limits the MSL complex production.   The second chromosome-wide regulatory system in flies acts on an autosome, the heterochromatic 4th chromosome. This regulation is a balancing mechanism between at least two different proteins, the chromosome 4 specific protein painting of fourth (POF) and heterochromatin protein 1 (HP1). POF binds to nascent RNAs transcribed from the 4th chromosome and HP1 target the same set of genes at the chromatin level. POF stimulates the transcribed genes, while HP1 represses them; together they create the most optimal condition for these genes. This type of balancing mechanism may be a more general way to fine-tune transcription at a chromosome-wide level and raises the question about autosomal gene regulation as a general mechanism.

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-26Bitstream added on 2014-06-13T19:12:50Z : No. of bitstreams: 1 bardella_vb_me_sjrp.pdf: 973541 bytes, checksum: 2642d082f6fe5ee2cb77ab3b60832684 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Os heterópteros apresentam a meiose cística nos túbulos seminíferos. Esses possuem o cisto espermatogonial envolto pelas células císticas, as quais desenvolvem a função de nutrição das células em divisão celular. Quanto às características citogenéticas, esses insetos apresentam cromossomos holocinéticos, baixa variabilidade cariotípica e meiose invertida dos cromossomos sexuais. No presente trabalho foram caracterizadas as células císticas quanto a sua localização, ultraestrutura e citogenética e, também, foram analisados os aspectos citogenéticos de quatro espécies do gênero Triatoma. Foram utilizadas as técnicas de microscopia eletrônica de transmissão, citogenética convencional (orceína e AgNOR), bandamento C CMA3/DAPI e a técnica de hibridização in situ fluorescente (FISH), com sonda de DNAr 45S de Drosophila melanogaster. Os resultados indicaram que a célula cística envolve um cisto espermatogonial e apresenta um grande núcleo com invaginações citoplasmáticas. Em todas as espécies foram observados vários graus de ploidia da célula cística. Triatoma infestans e T. infestans melanosoma apresentaram vários blocos heterocromáticos com a periferia CMA3 + e o interior DAPI+. Associada às bordas dos blocos heterocromáticos foram observados os segmentos de DNAr 45S, além da presença de vários nucléolos em cada núcleo. Triatoma matogrossensis, T. rubrovaria e T. brasiliensis apresentaram apenas um bloco heterocromático com as mesmas características, com exceção de T. brasiliensis, que apresentou em algumas células vários blocos CMA3 + dispersos. Nessas espécies foi observado apenas um nucléolo com similaridade na localização dos sítios de DNAr. Quanto aos aspectos citogenéticos, todas as espécies apresentaram 2n = 20A + XY, com decréscimo do tamanho relativo dos cromossomos. Em T. infestans melanosoma os cromossomos foram... / Heteroptera, or true bugs, exhibit meiosis in their seminiferous tubules. They posses the spermatogonial cysts that are enclosed by cyst cells, which develop the nutritional function of the cells during cell division. In terms of cytogenetic characteristics, these insects possess holokinetic chromosomes, low karyotype variability, and inverted meiosis in the sex chromosomes. In this study, cyst cells from four species of the genus Triatoma were characterized by their location, superstructure, and cytogenetic makeup. Electronic transmission microscopy techniques were used, as well as conventional cytogenetic techniques (Orcein and AgNOR), C-banding with CMA3 and DAPI banding, and Fluorescence in situ Hybridization (FISH) with a 45S DNA probe of Drosophila melanogaster. The results indicated that the the spermatogonial cyst is enclosed by the cyst cell, and that the cyst cell possesses a large nucleus with cytopasmic invaginations. In all species studied, varying degrees of ploidy were observed in the cyst cells. Triatoma infestans and T. infestans melanosoma presented with various heterochromatic blocks, with CMA3 + at the periphery and DAPI+ at the interior. Segments of rDNA 45S were found along the edges of the heterochromatic blocks, along with the presence of various nucleoli in each nucleus. Triatoma matogrossensis, T. rubrovaria and T. brasiliensis presented with only one heterochromatic block with the same characteristics (with the exception of T. brasiliensis, which presented with various dispersed CMA3 + blocks). In these species, only one nucleolus that was similar to the localization of the rDNA sites was found. All species presented with 2n = 20A + XY, with a decrease in size relative to the chromosomes. In the case of T. infestans melanosoma, the chromosomes were split into groups based on their relative sizes. The heterochromatin of this species presented... (Complete abstract click electronic access below)

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Agradecemos a compreensão. on 2017-10-26T16:14:49Z (GMT) / Submitted by MARIANI CRISTINA ALVES PICCOLI null (marianicpiccoli@gmail.com) on 2017-10-26T18:42:01Z No. of bitstreams: 1 versaofinalfinal.pdf: 2319438 bytes, checksum: 6f431d0bb6380c6dd4298885b7f2a00b (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-10-26T18:53:20Z (GMT) No. of bitstreams: 1 piccoli_mca_me_rcla.pdf: 2319438 bytes, checksum: 6f431d0bb6380c6dd4298885b7f2a00b (MD5) / Made available in DSpace on 2017-10-26T18:53:20Z (GMT). No. of bitstreams: 1 piccoli_mca_me_rcla.pdf: 2319438 bytes, checksum: 6f431d0bb6380c6dd4298885b7f2a00b (MD5) Previous issue date: 2017-08-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os DNAs repetitivos representam uma porção significante do genoma e podem estar envolvidos com os processos de variação cromossômica e reorganização genômica. Estes DNAs podem ser organizados em tandem ou dispersos, apresentando ampla variabilidade de composição e localização, sendo em muitos casos os principais constituintes da heterocromatina. A utilização dessas sequências como marcadores para a análise da evolução cromossômica é ampla em várias ordens de insetos, enquanto que para as abelhas esse conhecimento ainda é bastante restrito. Na tribo Meliponini o gênero Melipona se encontra dividido em quatro subgrupos e é o mais caracterizado cromossomicamente, sendo as espécies do gênero divididas ainda em dois grupos que apresentam marcante diferença na distribuição da heterocromatina. Um grupo é representado por espécies com pouca heterocromatina (principalmente centromérica) enquanto no segundo grupo as espécies apresentam heterocromatina dispersa ao longo de todos os cromossomos. Sendo assim, este gênero apresenta-se como um potencial modelo para estudos de amplificação e diversificação de DNAs repetitivos e sequências heterocromáticas. Neste contexto, utilizamos a espécie Melipona scutellaris que possui grande quantidade de heterocromatina nos cromossomos como modelo, buscando entender possíveis causas e eventos evolutivos envolvidos na ampla diferenciação heterocromática. Foram mapeadas distintas sequências de DNAs repetitivos por FISH, tais como DNAr 18S, DNAsn U2 e microssatélites, todos com localização eucromática, sugerindo que as sequências testadas não estão envolvidas com a amplificação da heterocromatina. A fração C0t-DNA e DOP-PCR evidenciou que a heterocromatina é composta por sequências altamente repetitivas, enquanto que a hibridização em membrana dessa fração em outras Meliponas demonstrou que a heterocromatina dentro do gênero difere de acordo com cada subgrupo. / Repetitive DNAs represent a significant portion of the genome and may be involved in the processes of chromosome variation and genomic rearrangement. These DNAs can be organized in tandem or dispersed, presenting wide variety of composition and location, being in many cases the main constituents of heterochromatin. An application as sequences as markers for an analysis of chromosomal evolution is broad in several orders of insects, whereas for bees this knowledge is still quite restricted. In the Meliponini tribe, the genus Melipona is divided into four subgroups and is the most characteristic chromosomally, being as species of the genus divided into two groups that present a marked differential in the distribution of heterochromatin. One group is represented by species with little heterochromatin (mainly centromeric) while not second group as species presented in heterochromatin dispersed throughout all the chromosomes. Thus, this genus presents itself as a potential model for amplification and diversification studies of repetitive DNAs and heterochromatic sequences. In this context, it uses a species Melipona scutellaris that has large amount of heterochromatin in the chromosomes as model, seeking to understand possible evolutionary events involved in the wide heterochromatic differentiation. Different FISH repeating DNA sequences have been mapped, such as 18S rDNA, U2 DNAs and microsatellites, all with euchromatic location, suggesting that as tested sequences are not involved with heterochromatin amplification. The C0t-DNA and DOP-PCR fraction showed that heterochromatin is composed of highly repetitive sequences, whereas a membrane hybridization of this fraction in other Meliponas demonstrated that heterochromatin within the genus differs according to each subgroup.

Orientador: Luciana Bolsoni Lourenço Morandini / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T00:03:54Z (GMT). No. of bitstreams: 1 Nascimento_Juliana_M.pdf: 2159511 bytes, checksum: 592ca4a405512c2a06472a0c276eb206 (MD5) Previous issue date: 2010 / Resumo: Os primeiros resultados citogenéticos obtidos para a espécie Physalaemus ephippifer, atualmente alocada no grupo P. cuvieri, mostraram um interessante heteromorfismo cromossômico ligado ao sexo. As 14 fêmeas analisadas presentaram um par cromossômico 8 heteromórfico, enquanto nos 7 machos analisados esse par era homomórfico. A diferença entre os cromossomos das fêmeas era devida à presença de um segmento adicional, composto por uma NOR e uma banda heterocromática a ela adjacente, localizado na região terminal do braço curto de apenas um desses homólogos, denominado de morfo 8b. Apesar desse importante achado, o uso de técnicas citogenéticas convencionais nessa e em outras espécies do grupo P. cuvieri não foram suficientes para esclarecer os mecanismos envolvidos na evolução cromossômica, já que poucos caracteres citogenéticos informativos foram evidenciados e também uma grande variação em relação às NORs foi encontrada. Com a intenção de buscar novos marcadores citogenéticos que auxiliem no estudo dos cromossomos sexuais de P. ephippifer e que colaborem na análise citogenética desse grupo de Physalaemus, foram estudadas sequências de DNA repetitivo isoladas desta espécie. Para tanto, o DNA genômico extraído de duas fêmeas de P. ephippifer, provenientes de Belém (Pará), foi digerido com a enzima de restrição BamHI e os fragmentos gerados foram separados por eletroforese em gel de agarose. Fragmentos posteriormente denominados de Pep194, Pep165 e Pep320 isolados a partir desse gel, foram clonados, sequenciados e localizados in situ no cariótipo de P. ephippifer. A seqüência Pep320 mostrou correspondência parcial com o fragmento EU343727.1 isolado de P. cuvieri, cuja sequência está disponível no GenBank. Apesar desses fragmentos não apresentarem similaridade entre si, as sequências Pep165 e Pep320 foram mapeadas no mesmo sítio cromossômico, correspondente à banda pericentromérica do braço curto do cromossomo 3. Esse resultado levanta um questionamento, ainda não respondido, acerca da organização molecular dessas sequências, que podem estar arranjadas em clusters independentes ou representar partes de uma mesma unidade repetitiva. A sequência Pep194 foi mapeada em regiões coincidentes com as NORs, localizadas no braço longo dos cromossomos identificados como Z e W, e no braço curto do cromossomo W. Apesar deste resultado, a análise da sequência Pep194 não apresentou nenhuma similaridade com regiões codificadoras do DNAr nucleolar, nem mesmo com regiões intergênicas associadas a elas já descritas. Tal sequência apresentou interessante arranjo interno, sendo composta de duas repetições diretas terminais, cada uma com 63 pb, sendo ambas flanqueadoras deuma região interna de 68 pb. Para melhor investigar a organização desta sequência no genoma de P. ephippifer, foram analisados produtos resultantes da amplificação por PCR de segmentos do DNA genômico, efetuada com o auxílio de primers construídos especificamente para se anelarem em regiões internas do segmento isolado. Os resultados obtidos por essa análise evidenciaram a presença de uma unidade repetitiva de 131 pb, que representa parte do fragmento Pep194, diferindo desse por não apresentar uma das regiões de 63 pb. No entanto, não é possível descartar a co-existência de uma unidade repetitiva de 194 pb, não detectada nessas análises. Em paralelo a esses experimentos, sequências pertencentes a elementos retrotransponíveis Rex1 foram isoladas do genoma de P. ephippifer por PCR, clonadas, sequenciadas e mapeadas por FISH em uma região heterocromática pericentromérica do braço curto do cromossomo 3. A análise das sequências desses fragmentos comprovou serem correspondentes a parte da sequência codificadora da enzima transcriptase reversa do elemento Rex1. A fim de verificar a presença desse elemento em outras espécies de Physalaemus, os mesmos primers utilizados nos experimentos com P. ephippifer, foram usados para a amplificação de sequências a partir de amostras do DNA genômico de P. albifrons, P. albonotatus, P. henselli e P. spiniger. A sequência isolada de P. albonotatus apresentou uma deleção interna de cerca de 220 pb quando comparada com as sequências correspondentes, aqui descritas ou disponíveis no GenBank. Isso permite sugerir que, embora derivado de um elemento Rex1, esse segmento provavelmente deixou de ser um elemento de transposição ativo. Embora esse seja o primeiro trabalho que descreve a ocorrência de Rex1 em anuros, a presença desse elemento parece comum, pelo menos no gênero Physalaemus. Além de apresentar similaridade com o segmento de Rex1 e com os fragmentos Pep165 e Pep320, a banda heterocromática pericentromérica de 3p é também o sítio de ocorrência de DNAr 5S. Tal região, reconhecida como DAPI-positiva na presente análise, permite clara distinção entre os cromossomos 3 e 4 de P. ephippifer, frequentemente confundidos se analisados apenas em relação à sua morfologia. As quatro sequências repetitivas aqui isoladas apresentaram-se eficientes marcadores citogenéticos e poderão ser utilizadas para futuros estudos comparativos entre espécies do gênero Physalaemus. / Abstract: Previous cytogenetic studies of Physalaemus ephippifer, a species currently allocated to the group P. cuvieri, showed an interesting female-specific chromosome heteromorphism. In 14 females of P. ephippifer, chromosome pair 8 was heteromorphic with regard to the occurrence of an NOR anda terminal C-band. Such heteromorphism was not found in the seven analyzed male specimens. These findings suggest that the chromosomes of pair 8 may be sex chromosomes in P. ephippifer, characterizing a ZZ ?/ ZW ? sex-determination system in this species. Despite these important data, conventional cytogenetic techniques performed in this and other species of the Physalaemus group were not sufficient to clarify the processes involved in the karyological divergence in this anuran group. Aiming to look for new cytogenetic markers that may help in the study of P. ephippifer sex chromosomes and in the cytogenetic analysis of this group of Physalaemus, we studied repetitive DNA sequences isolated from P. ephippifer. Genomic DNA extracted from two females of P. ephippifer from Belém (Pará) was digested with the restriction enzyme BamHI. The restriction fragments were separated by electrophoresis in agarose gel. DNA fragments, which were ultimately named Pep194, Pep165, and Pep320, were isolated from the gel, cloned, sequenced, and localized in situ in the karyotype of P. ephippifer. The sequence Pep320 was very similar to the fragment EU343727.1 isolated from P. cuvieri. Although Pep320 and Pep165 were totally different in nucleotide sequence, they were mapped on the same chromosome site, which corresponded to the pericentromeric C-band in the short arm of chromosome 3. This raises doubts about the molecular organization of these sequences, which can be arranged in independent clusters, but can also represent partial regions of the same repeat unit. The sequence Pep194 was mapped in regions that coincided with the NORs, located in the long arm of Z and W chromosomes and in the short arm of the W chromosome. However, the Pep194 sequence had no similarity with the coding regions of the nucleolar rDNA or with intergenic spacers associated to them. The restriction fragment Pep194 had an interesting internal arrangement, being composed of two terminal direct repeats, each with 63 bp, flanking an internal region of 68 bp. To further investigate the organization of this sequence in the genome of P. ephippifer, we amplified some Pep194 segments from genomic DNA by PCR using specific primers designed to anneal in inner / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

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No. of bitstreams: 1 Dissertação - Sabrina Emanuela de Melo Araújo.pdf: 1064742 bytes, checksum: bea4234c7d69e414fb2af0d8af97b7cf (MD5) Previous issue date: 2016-02-17 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Among the Chiropteran, the Phyllostomidae family is the most diverse clade of the Neotropics and in Amazon are found about 80 species. From a chromosomal point of view, Phyllostomidae stands out for presenting many karyotype variation, with diploid numbers ranging from 2n = 14 in Vampyressa melissa (Stenodermatinae) to 2n = 46 in Macrotus waterhousii (Macrotinae). There are several genetic mechanisms or processes that can result in numeric/structural chromosomal abnormalities and often repetitive DNA sequences are involved in this process. In order to understand the variety and karyotype evolution of this family, classical and molecular cytogenetic analyzes were performed on mitotic chromosomes of four species belonging to four subfamilies of distinct phylogenetic clades: Artibeus obscurus, Carollia perspicillata, Desmodus rotundus and Phylostomus elongatus. Artibeus obscurus presented NF = 56 and 2 N = 30/31, with 22m + 6st + XX / XY1Y2; C. perspicillata NF = 36 and 2N = 20/21, with 14m-sm + 4st + XX / XY1Y2; D. rotundus NF = 52 and 2 N = 28, and 26m-sm + XX / XY; P. elongatus NF = 60 and 2 N = 32, and 28m-sm + 2a + XX / XY. The distribution pattern of constitutive heterochromatin revealed a signal in the pericentromeric region in all chromosomes of the four analyzed species and intraspecific variations were observed when compared the results of this work with the one in existing literature. Regarding the signal of ribosomal DNA sites 18S and nucleolus organizer regions active in A. obscurus were shown in the terminal region of the pairs 5, 6 and 7; C. perspicillata in pericentromeric region of the X chromosome; D. rotundus in the centromeric region of pair 8 and in P. elongatus in the centromeric/terminal region of the pair 15. Conspicuous telomeric signals were observed in D. rotundus and P. elongatus, while in A. obscurus and C. perspicillata the terminals signals are blurred. Interstitial telomeric sites were absent in P. elongatus and present in other species, which may indicate mergers. The LINE-1 retroelement presented scattered signals, however in some chromosomes they are identical to the patterns of dark bands evident in the band G and is also accumulated on chromosome X. If compared the phylogenetic position of the studied species, it is noted that the most derived taxons accumulate high karyotype variation as repetitive elements. / Dentre os Chiropteros, a família Phyllostomidae constitui o clado mais diversificado do neotrópico e na Amazônia são encontradas cerca de 80 espécies. Do ponto de vista cromossômico, Phyllostomidae destaca-se por apresentar grande variação cariotípica, com números diplóides que vão de 2n=14 em Vampyressa melissa (Stenodermatinae) a 2n=46 em Macrotus waterhousii (Macrotinae). São vários os processos ou mecanismos genéticos que podem resultar em alterações cromossômicas numéricas/estruturais e muitas vezes sequências repetitivas de DNA estão envolvidas neste processo. Visando compreender a variedade e a evolução cariotípica desta família, foram realizadas análises citogenéticas clássicas e moleculares em cromossomos mitóticos de quatro espécies, pertencentes a quatro subfamílias de clados filogenéticos distintos: Artibeus obscurus, Carollia perspicillata, Desmodus rotundus e Phylostomus elongatus. Artibeus obscurus apresentou NF=56 e 2N=30/31, sendo 22m+6st+XX/XY1Y2; C. perspicillata NF=36 e 2N=20/21, sendo 14m-sm+4st+XX/XY1Y2; D. rotundus NF=52 e 2N=28, sendo 26m-sm+XX/XY; P. elongatus NF=60 e 2N=32, sendo 28m-sm+2a+XX/XY. O padrão de distribuição da heterocromatina constitutiva revelou marcação na região pericentromérica em todos os cromossomos das quatro espécies analisadas e variações intraespecíficas foram observadas quando comparados os resultados deste trabalho com o existente na literatura. Em relação à marcação de sítios de DNA ribossomal 18S e regiões organizadoras de nucléolo ativas, em A. obscurus foram evidenciados na região terminal dos pares 5, 6 e 7; em C. perspicillata na região pericentromérica do cromossomo X; em D. rotundus na região centromérica do par 8 e em P. elongatus na região centromérica/terminal do par 15. Marcações teloméricas conspícuas foram visualizadas em D. rotundus e P. elongatus, enquanto que em A. obscurus e C. perspicillata as marcações terminais são tênues. Sítios teloméricos intersticiais foram ausentes em P. elongatus e presente nas demais espécies, podendo ser indicativo de fusões. O retroelemento LINE-1 revelou marcação dispersas, porém em alguns cromossomos são coincidentes com os padrões de bandas escuras evidenciados na banda G, sendo também acumulados no cromossomo X. Se comparada a posição filogenética as espécies estudadas, nota-se que os táxons mais derivados, acumulam maior variação cariotípica, assim como os elementos repetitivos.