DNA topological stress impedes normal DNA replication. If topological stress is allowed to build up in front of the replication fork, the fork rotates to overcome the stress, leading to formation of DNA pre-catenanes. The formation of DNA pre-catenanes is therefore a marker of DNA topological stress. In this study, I have examined how transcription linked DNA topological stress impacts on fork rotation and on endogenous DNA damage. Transcription, similar to replication, affects the topology of the DNA; and collision between the two machineries is likely to lead to high levels of DNA topological stress. I found that the frequency of fork rotation during DNA replication, increases with the number of genes present on a plasmid. Interestingly, I also found that this increase in pre-catenation is dependent on the cohesin complex. Cohesin and transcription are known to be linked, as transcription leads to the translocation of cohesin along budding yeast DNA away from its loading sites. Cohesin plays a major role in establishing chromosomal structure, influencing gene expression and genetic inheritance. In this work, I have analysed the relationship between cohesin and the generation of topological stress and found that topological stress associated with cohesin can lead to DNA replication stress and DNA damage.

Orientador: José Osmar Gaspar / Banca: Claudia Regina Bonini Domingos / Banca: Eliezer Rodrigues de Souto / Resumo: O presente trabalho consistiu no seqüenciamento e caracterização molecular das Cadeias Abertas de Leitura (Open Reading Frames - ORFs) 2 e 3 do genoma do isolado São Paulo do Southern bean mosaic virus (SBMV-SP), completando-se o seqüenciamento de todo o genoma desse isolado. A ORF 2 codifica uma poliproteína (serino protease - VPg - RNA polimerase RNA-dependente) e a ORF 3 um produto com função desconhecida. O seqüenciamento da ORF 2 apresentou 2889 nucleotídeos, incluindo-se o códon de terminação UGA, com 962 aminoácidos deduzidos e massa molecular estimada de aproximadamente 105 kDa. Dentro da ORF 2, localiza-se a ORF 3 contendo 398 nucleotídeos, incluindo-se o códon de terminação UAA, com 132 aminoácidos e massa molecular estimada de aproximadamente 15 KDa. A análise feita a partir das seqüências das ORFS 2 e 3 do genoma do SBMV-SP, quando comparadas com outras espécies do mesmo gênero e isolados do SBMV, depositadas no GenBank, mostrou que a ORF 2 apresenta maior identidade (91,4% na seqüência de nucleotídeos e 95,0% na seqüência de aminoácidos deduzidos) com o isolado de Arkansas. Resultado similar foi obtido em relação à ORF 3 com valores de identidade de 97,0% tanto para as seqüências de nucleotídeos e aminoácidos deduzidos. Dados de filogenia corroboram os dados de identidade. Regiões conservadas do gênero Sobemovirus também foram identificadas, tais como Sítio de Ligação à Capa Protéica (CBPS), a tríade catalítica da serino protease (H-D-S) e a seqüência de heptanucleotídeos (TTTAAAC). A expressão em Escherichia coli da porção C-terminal da RNA Polimerase RNA Dependente (RpRd) produziu uma proteína de fusão de aproximadamente 67 kDa no sistema pMAL c2-x e de 30 kDa no sistema pET 28a. Quando a proteína de fusão foi injetada em coelhos houve a produção de anti-soro específico para a proteína recombinante. / Abstract: The present work consisted of the sequencing and molecular characterization of the Open Reading Frames (ORFs) 2 and 3 from the São Paulo isolate genome of Southern bean mosaic virus (SBMV-SP), completing the sequencing of all the genome of this isolate. The ORF 2 encodes a polyprotein (serine protease - VPg - RNA dependent RNA polimarase) and the ORF 3 one product with unknown function. The sequencing of the ORF 2 reveals 2889 nucleotides, including the stop codon (UGA), with 962 deduced amino acids e and estimated molecular weight of approximately 105 kDa. Nested in the ORF 2 were found the ORF 3 with 398 nucleotides, including the stop codon (UAA), with 132 deduced amino acids and estimated molecular weight of approximately 15 kDa. The analysis made from the sequences of the ORFs 2 and 3 from the SBMV-SP genome, when compared with other species of the same gender and isolates of SBMV, deposited in the GenBank, showed that the ORF 2 presents higher identity (91,4% in the nucleotide sequence and 95,0% in the deduced amino acids sequence) with Arkansas isolate. Similar result was obtained in relation to the ORF 3 with identity values of 97,0% for the nucleotides and deduced amino acids sequences. Phylogeny data corroborate the identity data. Conserved regions of the Sobemovirus gender had also been identified such as Coat Protein Binding Site (CPBS), serine protease catalytic triad (H-D-S) and heptanucleotide sequence (TTTAAAC). The Expression in Escherichia coli of the C-terminal region of the RNA dependent RNA polimerase produced a fusion protein of approximately 67 kDa in pMAL c2-x system and a 30 kDa protein in pET 28a system. When the fusion protein ...(Complete abstract click electronic access below) / Mestre

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Introduction The present study evaluated reported methodological characteristics of GWAS, investigating relationships between reported methodological characteristics and outcomes observed. Methods GWAS were identified from NHGRI’s catalogue of GWAS (2005 to 2009). Multivariate meta-regression models (random effects) were produced to identify the impact of reported study characteristics and the strength of relationships between the variables and outcomes. Results The summary odds ratios for replication components of GWAS in cancer was 1.34 (95% CI 1.25, 1.43) and neuropsychiatric disorders was 1.43 (95% CI 1.30, 1.57). Heterogeneity was accounted for by nature of the control group, relationship between case/control groups, whether cases/controls were drawn from the same population, if data was a primary collection or a build on pre-existing data, if quality assurance was reported, and if the study reported power/sample size. Conclusion Evidence supports the existence of variability in reporting, with index components demonstrating less variability than replication components in the GWAS.

When Darwin introduced natural selection in 1859 as a key mechanism of evolution, little was known about the underlying cause of variation within a species. Today we know that this variation is caused by the acquired genomic differences between individuals. Polymorphism, defined as the existence of multiple alleles or forms at a genomic locus, is the technical term used for such genetic variations. Polymorphism, along with reproduction and inheritance of genetic traits, is a necessary condition for natural selection and is crucial in understanding how species evolve and adapt. Many questions regarding polymorphism, such as why certain species are more polymorphic than others or how different organisms tend to favor some types of polymorphism among others, when solved, have the potential to shed light on important problems in human medicine and disease research. Some of these studies require more diverse species and/or individuals to be sequenced. Of particular interest are species with the highest rates of polymorphisms. For instance, the sequencing of the sea squirt genome lead to exciting studies that would not be possible to conduct on species that possess lower levels of polymorphism. Such studies form the motivation of this thesis. Sequencing of genomes is, nonetheless, subject to its own research. Recent advances in DNA sequencing technology enabled researchers to lead an unprecedented amount of sequencing projects. These improvements in cost and abundance of sequencing revived a greater interest in advancing the algorithms and tools used for genome assembly. A majority of these tools, however, have no or little support for highly polymorphic genomes; which, we believe, require specialized methods. In this thesis, we look at challenges imposed by polymorphism on genome assembly and develop methods for polymorphic genome assembly via an overview of current and past methods. Though we borrow fundamental ideas from the literature, we introduce several novel concepts that can be useful not only for assembly of highly polymorphic genomes but also genome assembly and analysis in general.

Introduction The present study evaluated reported methodological characteristics of GWAS, investigating relationships between reported methodological characteristics and outcomes observed. Methods GWAS were identified from NHGRI’s catalogue of GWAS (2005 to 2009). Multivariate meta-regression models (random effects) were produced to identify the impact of reported study characteristics and the strength of relationships between the variables and outcomes. Results The summary odds ratios for replication components of GWAS in cancer was 1.34 (95% CI 1.25, 1.43) and neuropsychiatric disorders was 1.43 (95% CI 1.30, 1.57). Heterogeneity was accounted for by nature of the control group, relationship between case/control groups, whether cases/controls were drawn from the same population, if data was a primary collection or a build on pre-existing data, if quality assurance was reported, and if the study reported power/sample size. Conclusion Evidence supports the existence of variability in reporting, with index components demonstrating less variability than replication components in the GWAS.

Microorganisms are a goidmine for evolutionary genetics as their genomes can evolve at an extraordinary rate which results in some of the most extravagant adaptations in terms of genome structure and function as well as survival in the most unusual environments. One trend observed in several evolutionary scenarios is genome degeneration. It is most prominent in endosymbionts and obligate intracellular parasites and is a consequence of many constraints encountered in the intracellular environment. The process involves loss of many protein-coding genes, resulting in greater dependence on the host, and loss of non-coding DNA such as intergenic regions, which has a direct impact on regulation of genome function. I have chosen two evolutionarily distinct systems to analyze the stages and functional consequences of genome degeneration, namely the impact of genome compression on transcription in an obligate parasite Antonospora locustae (genus Microsporidia), and gene content in the mitochondrion of a diatom endosymbiont found in the dinoflagellate Durinskia baltica. I have successfully mapped transcriptional start and termination sites from 14 loci in Antonospora locustae, and cloned fragments of two genes that are part of the electron transport chain from the mitochondrion of the diatom endosymbiont in Durinskia baltica. My analysis reveals that transcription in A. locustae is always initiated immediately upstream of the open reading frame at a single point for every locus, whereas transcriptional termination can occur at several points for a single gene and, in some instances overlaps with a downstream reading frame. The identification ofNADH5 and ATPase9 from the mitochondrion of the endosymbiont in D. baltica is further evidence for the preservation of function in this enigmatic organelle.

Telomeres comprise the physical ends of chromosomes. Essential functions of telomeres include protecting the terminus from being recognized as a DNA doublestrand break and facilitating the complete replication of the physical end of the DNA. Telomere functions are mediated by a large array of telomere-associated proteins. Mutations in telomere-related genes cause immediate telomere dysfunction, activation of DNA damage response, and accumulation of end-to-end chromosome fusions. In addition, changes in telomere complex composition may affect the ability of the telomerase enzyme to maintain telomeres in vivo. Here, we describe the characterization of telomere-associated proteins in the flowering plant, Arabidopsis thaliana. Using a bioinformatics approach, we identified twelve proteins with sequence similarity to vertebrate duplex telomere DNA binding proteins TRF1 and TRF2. We showed that, like their vertebrate counterparts, some of the Arabidopsis TRFL (TRF-LIKE) proteins can homodimerize and bind telomeric DNA in vitro, indicating that Arabidopsis encodes a large family of double-strand telomeric DNA binding proteins. We have also characterized three Arabidopsis POT1 proteins whose homologs in yeast and vertebrates associate with the single-stranded portion of telomeric DNA. Unexpectedly, we found that unlike POT1 protein in other organisms, Arabidopsis AtPOT1a protein associates with telomeres only in the S phase of the cell cycle and is a physical component of the active telomerase RNP complex, providing positive telomere length regulation. Our data implicated AtPOT1b, another Arabidopsis POT1 protein, in chromosome end protection. Finally, we showed that Arabidopsis thaliana has evolved a third POT1 protein, AtPOT1c, which contributes to both telomere length regulation and telomerase activity, and maintenance of the structure of the chromosome terminus. Thus, Arabidopsis has evolved a set of POT1 proteins that make distinct and novel contributions to telomere biology. Finally, we describe the identification and characterization of a novel Arabidopsis protein CIT1 (Critical for Integrity of Telomeres 1), and show that CIT1 deficiency leads to an immediate and profound telomere dysfunction and chromosome end deprotection. Altogether, these data provide new insight into plant telomereassociated factors and significantly improve our understanding of the overall architecture and evolution of telomeric complex in Arabidopsis.

Waves of bovine genomic data have been produced as a result of the bovine genome sequencing projects. In addition to the massive amounts of genomic sequence, significant annotation including single nucleotide polymorphisms, sequence tagged sites and haplotype blocks have been produced by the Bovine HapMap Project. Furthermore, many agriculturally significant traits in cattle such as milk yield and carcass weight are measured on a quantitative scale and have been genetically mapped as quantitative trait loci (QTL). QTL data can be used to generate another form of bovine annotation linking phenotype to genotype. However, it is impossible for humans to be able to analyze genomic scale data without computer based tools. Bioinformatic tools have been shown to greatly increase productivity and improve efficiency when dealing with large data sets. My dissertation presents an integrated, extensible database that houses SNPs, STSs, haplotypes, and QTL. The database is presented to researchers through a restructured, object oriented Bovine QTL Viewer that displays multiple levels of bovine annotation synergistically. Evaluation of use of the viewer was performed using a survey based approach and measured quantitatively. In addition, the QTL data from the database was used to analyze the frequency of gene ontology (GO) annotations within QTL regions. QTL regions were divided into 8 trait based groups. GO terms were counted within each category of QTL and in non- QTL regions of the genome. Top level GO term frequencies were generated from the counts and these frequencies were compared between QTL and non-QTL portions of the genome. Furthermore, specific sets of GO terms believed to be related to QTL categories were also used to determine if QTL regions were enriched for genes annotated with such GO terms. As a result, we determined that gene density varied significantly across QTL regions and that many QTL categories showed GO term frequency differences that could be related to the trait’s biology. Furthermore, our selected GO term sets were shown to be significantly enriched in some QTL categories.

Thesis (Ph. D.)--University of Oregon, 2000. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-86).