G-quadruplexes, a type of nucleic acid secondary structure consisting largely of folded quartets of guanines, appear to play a regulatory role in the human genome. Heme has been shown to interact with G-quadruplexes. The ChIP-Seq-like Heme-Seq assay was developed to identify heme binding G-quadruplex loci. Using Heme-Seq, 3 primary heme binding loci and 4 secondary minor heme binding loci were identified on six chromosomes. Two of the primary heme binding loci were found at the centromeric boundaries of the long arms of metacentric chromosomes with the majority of reads from the primary heme binding loci consisting primarly of Human Satellite II (HSATII) nucleotide repeat sequences. Numerous putative G-quadruplex forming sequences were found in the heme-binding locus on Chromosome 2. Comparison of Heme-Seq results with available data from a G-quadruplex ChIP-Seq study in live cells, revealed that the regions which exhibited binding at the three peaks from the Heme-Seq data also showed binding coverage in the CHIP-Seq data. In addition to the known association with G-quadruplexes, heme also appears to bind to HSATII repeats,. The biological role and importance of this binding is not known. / Biomedical Sciences

Repetitive tetramers of the DNA sequence 5'-CAAT-3' are present in several loci associated with lipooligosaccharide (LOS) phase variation in Haemophilus influenzae type b (Hib). In an attempt to identify H. somnus phase-variable LOS genes, the presence of CAAT repeats within the H. somnus 738 genome was confirmed using a (CAAT)7 probe. A 3.9 kb EcoRI fragment that reacted with the probe was cloned and sequenced. Sequence analysis confirmed the presence of 31 CAAT repeats downstream of two potential start codons, and indicated that small or large proteins would be encoded depending on the number of CAAT repeats. The larger gene products showed 46% amino acid homology to Lex2b from Hib, which influences LOS phase variation in that species. In H. somnus, this gene was named lob1 (lipooligosaccharide biosynthesis gene). Sequence analysis showed that randomly selected colonies most frequently contained 33 CAAT repeats in lob1, corresponding to a 294 amino acid product. Colonies selected for negative reactivity to mAb 5F5 were significantly more likely to have different numbers of CAAT repeats in lob1 than randomly selected colonies. The presence of lob1 in trans altered the LOS profile of a non-phase variable strain of H. somnus, and caused increased levels of reactivity to polyclonal antisera made to purified LOS from strain 738. Based on the ability of this gene to alter the LOS profile of a non-phase varying strain and the correlation of changes in CAAT repeats with mAb 5F5 reactivity, lob1 appears to be involved in LOS biosynthesis and phase variation. / Master of Science

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-16T21:36:49Z No. of bitstreams: 1 Enio Paulo Estimativa de mistura étnica avaliada por Marcadores Informativos de.pdf: 352598 bytes, checksum: 7d448dc54afe1ec271f59fc912275f41 (MD5) / Made available in DSpace on 2012-07-16T21:36:49Z (GMT). No. of bitstreams: 1 Enio Paulo Estimativa de mistura étnica avaliada por Marcadores Informativos de.pdf: 352598 bytes, checksum: 7d448dc54afe1ec271f59fc912275f41 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A miscigenação entre os três principais grupos étnicos (ameríndios, europeus e africanos) originou a alta diversidade genética da população brasileira. Na Bahia a proporção de afrodescendentes é de 77,5%, sendo que em Salvador 79,8% se auto-denominam negros ou pardos. Poucos estudos descrevem a diversidade genética da população baiana e a contribuição de cada grupo étnico na sua formação. Diversos marcadores de DNA são atualmente utilizados para estimar mistura étnica em populações miscigenadas. Estes marcadores são denominados alelos específicos de população (PSAs) ou marcadores informativos de ancestralidade (AIMs) e apresentam alelos com grandes diferenciais de freqüência, superiores a 30%, entre populações geográfica ou etnicamente definidas. Os microssatélites (STRs) são variantes genéticos úteis no mapeamento genético de espécies, na identificação de pessoas, mapeamento genético e análise de populações. Alguns STRs apresentam alelos com freqüências marcantes em determinados grupos populacionais. Com objetivo de comparar a ancestralidade genomica avaliada com dois tipos de marcadores, foram estudados 8 microssatélites STRs autossômicos (TH01, vWA31, D18S51, FGA, TPOX, D7S820, D3S1358, D8S1179) e 9 AIMs (FY-Null, LPL, AT3-I/D, Sb19.3, APO, PV92, CYP3A4, CKMM, GC-1F e GC-1S), em 203 indivíduos miscigenados da Bahia. A genotipagem foi realizada por PCR (Polimerase Chain Reaction), para deleções, inserções e para os microssatélites e PCR quantitativo em tempo real para mutações pontuais. As contribuições africana, européia e ameríndia observadas foram respectivamente 33,5%, 58,6% e 7,9% para os STRs e 45,08%, 45,16% e 9,75% para os AIMs, comprovando a miscigenação da população. O Índice Kappa, mostrou que a concordância entre as estimativas de ancestralidade utilizando os dois tipos de marcadores (AIMs e STRs), foi muito baixa (kappa = 0,12). Foi observada associação entre sobrenome de conotação religiosa e ancestralidade africana / The mixing between the three main ethnic groups (Amerindians, Europeans and Africans) produced a high genetic diversity of the braziliam population. In Bahia, the proportion of African descent that call themselves black or brown is 77.5% and 79.8% in Salvador. Few studies describe the genetic diversity of the population of Bahia and the contribution of each ethnic group in its formation. Several DNA markers are currently used to estimate ethnic mix in admixed populations. These markers are called alleles specific population (PSAs) or ancestry informative markers (AIMs) and carry alleles with large differences in frequency above 30% between populations geographically or ethnically defined. Microsatellites (STRs) are useful genetic variants in the genetic mapping of species, identification of persons, genetic mapping and analysis of populations. Some STRs have alleles with frequencies marked in certain population groups. To compare the ancestry genomica evaluated with two types of markers were studied 8 microsatellite autosomal STRs (TH01, vWA31, D18S51, FGA, TPOX, D7S820, D3S1358, D8S1179) and 9 AIMs (FY-Null, LPL, AT3-I /D, Sb19.3, APO, PV92, CYP3A4, CK-MM, GC and GC-1F-1S) in 203 subjects with mixed Bahia. Genotyping was performed by PCR (Polymerase Chain Reaction), for deletions, insertions and for microsatellite and quantitative PCR in real time for mutations. The contributions of African, European and Amerindian observed were respectively 33.5%, 58.6% and 7.9% for the STRs and 45.08%, 45.16% and 9.75% for the AIMs, proving the mixing of population. The Kappa index showed that the correlation between the estimates of ancestry using both types of markers (AIMs and STRs), was very low (kappa = 0.12). Association was found between devotional surnames and African ancestry.

No description available.

BACKGROUND: Comparative evolutionary analysis of whole genomes requires not only accurate annotation of gene space, but also proper annotation of the repetitive fraction which is often the largest component of most if not all genomes larger than 50 kb in size. RESULTS: Here we present the Rice TE database (RiTE-db) - a genus-wide collection of transposable elements and repeated sequences across 11 diploid species of the genus Oryza and the closely-related out-group Leersia perrieri. The database consists of more than 170,000 entries divided into three main types: (i) a classified and curated set of publicly-available repeated sequences, (ii) a set of consensus assemblies of highly-repetitive sequences obtained from genome sequencing surveys of 12 species; and (iii) a set of full-length TEs, identified and extracted from 12 whole genome assemblies. CONCLUSIONS: This is the first report of a repeat dataset that spans the majority of repeat variability within an entire genus, and one that includes complete elements as well as unassembled repeats. The database allows sequence browsing, downloading, and similarity searches. Because of the strategy adopted, the RiTE-db opens a new path to unprecedented direct comparative studies that span the entire nuclear repeat content of 15 million years of Oryza diversity.

Microsatellites are short (1-6bp long) highly polymorphic tandem repeats, found in all genomes analyzed so far. Popular genetic markers for many applications including population genetics, pedigree analysis, genetic mapping and linkage analysis, some microsatellites also can cause a variety of human neurodegenerative diseases and may act as agents of adaptive evolution through the regulation of gene expression. As a consequence of these diverse uses and functions, the mutational and evolutionary dynamics of microsatellite sequences have gained much attention in recent years. Mostly, the focus of studies investigating microsatellite evolution has been to develop more refined evolutionary models for estimating parameters such as genetic distance or linkage disequilibrium. However, there is an incentive in using our understanding of the evolutionary processes that affect these sequences to examine the functional implications of microsatellite evolution. What has emerged from nearly two decades of study are highly complex mutational dynamics, with mutation rates varying across species, loci and alleles, and a multitude of potential influences on these rates, most of which are not yet fully understood. The increasing availability of whole genome sequences has immensely extended the scope for studying microsatellite evolution. For example, where once it was common to examine single loci, it is now possible to examine microsatellites using genome wide approaches. In the first part of my dissertation I discuss approaches and issues associated with detecting microsatellites in genomic data. In Chapter 2 I undertook a meta-analysis of studies investigating the distribution of microsatellites in yeast and showed that studies comparing the distribution of microsatellites in genomic data can be fraught due to the application of different definitions for microsatellites by different investigators. In particular, I found that variation in how investigators choose the repeat unit size of a microsatellite, handle imperfections in the array and especially the choice of minimum array length used, leads to a large divergence in results and can distort the conclusions drawn from such studies, particularly where inter-specific comparisons are being made. In a review of the currently available suite of bioinformatics tools (Chapter 3), I further showed that this bias extends beyond a solely theoretical controversy into a methodological issue because most software tools not only incorporate different definitions for the key parameters used to define microsatellites, but also employ different strategies to search and filter for microsatellites in genomic data. In this chapter I provide an overview of the available tools and a practical guide to help other researchers choose the appropriate tool for their research purpose. In the second part of my thesis, I use the analytical framework developed from the previous chapters to explore the biological significance of microsatellites exploiting the well annotated genome of the model organism Saccharomyces cerevisiae (baker’s yeast). Several studies in different organisms have indicated spatial associations between microsatellites and individual genomic features, such as transposable elements, recombinational hotspots, GC-content or local substitution rate. In Chapter 4, I summarized these studies and tested some of the underlying hypotheses on microsatellite distribution in the yeast genome using Generalized Linear Models (GLM) and wavelet transformation. I found that microsatellite type and distribution within the genome is strongly governed by local sequence composition and negative selection in coding regions, and that microsatellite frequency is inversely correlated with SNP density reflecting the stabilizing effect point mutations have on microsatellites. Microsatellites may also be markers for recent genome modifications, due to their depletion in regions nearby LTR transposons, and elements of potential structural importance, since I found associations with features such as meiotic double strand breaks, regulatory sites and nucleosomes. Microsatellites are subject to local genomic influences, particularly on small (1-2kb) scales. Although, these local scale influences might not be as dominant as other factors on a genome-wide scale they are certainly of importance with respect to individual loci. Analysis of locus conservation across 40 related yeast strains (Chapter 5) showed no bias in the type of microsatellites conserved, only a negative influence of coding sequences, which supports again the idea that microsatellites evolve neutrally. Polymorphism was rare, and despite a positive correlation with array length, there was no relationship with either genomic fraction or repeat size. However, the analysis also revealed a non-random distribution of microsatellites in genes of functionally distinct groups. For example, conserved microsatellites (similar to general microsatellites in yeast) are mostly found in genes associated with the regulation of biological and cellular processes. Polymorphic loci show further an association with the organization and biogenesis of cellular components, morphogenesis, development of anatomical structures and pheromone response, which, is absent for monomorphic loci. Whether this distribution is an indication of functionality or simply neutral mutation (e.g. genetic hitch-hiking) is debatable since most conserved microsatellites, particularly variable loci, are located within genes that show low selective constraints. Overall, microsatellites appear as neutrally evolving sequences, but owing to the sheer number of loci within a single genome, individual loci may well acquire some functionality. More work is definitely needed in this area, particularly experimental studies, such as reporter-gene expression assays, to confirm phenotypic effects.

Um dos maiores desafios na área forense é, sem dúvida, a identificação humana. O DNA tem sido o responsável por uma verdadeira revolução das técnicas de identificação nas últimas décadas a partir do estudo e da identificação de polimorfismos entre determinados marcadores moleculares existentes nos indivíduos. Os marcadores recomendados para a obtenção de perfis genéticos são loci de regiões microssatélite do DNA, também designados de Short Tandem Repeats (STR). O número de repetições dos marcadores STR é variável entre os indivíduos, criando polimorfismo e tornando-os, desta forma, ótimos marcadores para identificação humana. O objetivo desse trabalho foi estabelecer a distribuição da frequência alélica de 16 STRs preconizados no sistema CODIS, na capital e no Departamento Central de Paraguai. Foram estudadas 300 amostras de saliva coletadas com NUCLEIC-CARD(TM) Collection Device System, de indivíduos paraguaios dentre 20 e 70 anos de idade, morando em uma das 20 cidades estudadas. Para o processamento foi utilizado o kit AmpFLSTR Identifiler Direct PCR Amplification® seguindo as fases de genotipagem, amplificação e eletroforese capilar. Foi possível estabelecer o perfil genético de 259 amostras bem como os parâmetros forenses e, assim, calcular os loci mais polimórficos os quais foram FGA e D18S51 utilizando os softwares GenAlEx 6.5, Arlequin 3.5 e R 2.5. A distribuição das frequências alélicas de cada loci analisado permitiu estabelecer a caracterização genética da população estudada. Foi possível confirmar que a população do Paraguai se encontra em equilíbrio Hardy-Weinberg, com uma diversidade genética intrapopulacional de 0.794915 +/- 0.398307 e interpopulacional determinada pelo índice de fixação (FST) de 0.01112. A partir desse estudo, foi possível determinar as frequências alélicas de 15 STRs utilizados no sistema CODIS nacapital e no Departamento Central do Paraguai bem como a caracterização genética e os parâmetros forenses da população estudada. Todos os loci estudados na população paraguaia foram considerados muito informativos e úteis para a solução de problemas relacionados com identificação humana na amostra analisada / One of the biggest challenges in Forensic Sciences is undoubtedly human identification. DNA has been responsible for a true revolution in identification techniques in the last decades from the study and identification of polymorphisms between certain molecular markers in individuals. The recommended markers for obtaining genetic profiles are loci of DNA microsatellite regions, also called Short Tandem Repeats (STR). The number of repetitions of STR markers is variable among individuals, creating polymorphism and thus making them excellent markers for human identification. The aim of this study was to establish the distribution of the allelic frequency of 16 STRs recommended in the CODIS system, in the capital and in the Central Department of Paraguay. We studied 300 saliva samples collected from NUCLEIC-CARD (TM) Collection Device System of Paraguayan individuals between 20 and 70 years-old, living in one of the 20 cities studied. For the processing, the AmpFLSTR Identifiler Direct PCR Amplification® kit was used following the phases of genotyping, amplification and capillary electrophoresis. It was possible to establish the genetic profile of 259 samples as well as the forensic parameters and thus to calculate the most polymorphic loci which were FGA and D18S51 using the software GenAlEx 6.5, Arlequin 3.5 and R 2.5. The distribution of the allelic frequencies of each analyzed loci allowed establishing the genetic characterization of the studied population. It was possible to confirm that the population of Paraguay is in Hardy-Weinberg equilibrium, with an intrapopulational genetic diversity of 0.8046 +/- 0.0120 and interpopulational determined by the fixation index (FST) of 0.01112. From this study, it was possible to determine the allelic frequencies of 15 STRs used in the CODIS system in the capital and in the Central Department of Paraguay, as well as the genetic characterization and forensic parameters of the studied population. All the loci studied in the Paraguayan population were considered veryinformative and useful for the solution of problems related to human identification in the analyzed sample

by Wai-keung Leung. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 217-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Um dos maiores desafios na área forense é, sem dúvida, a identificação humana. O DNA tem sido o responsável por uma verdadeira revolução das técnicas de identificação nas últimas décadas a partir do estudo e da identificação de polimorfismos entre determinados marcadores moleculares existentes nos indivíduos. Os marcadores recomendados para a obtenção de perfis genéticos são loci de regiões microssatélite do DNA, também designados de Short Tandem Repeats (STR). O número de repetições dos marcadores STR é variável entre os indivíduos, criando polimorfismo e tornando-os, desta forma, ótimos marcadores para identificação humana. O objetivo desse trabalho foi estabelecer a distribuição da frequência alélica de 16 STRs preconizados no sistema CODIS, na capital e no Departamento Central de Paraguai. Foram estudadas 300 amostras de saliva coletadas com NUCLEIC-CARD(TM) Collection Device System, de indivíduos paraguaios dentre 20 e 70 anos de idade, morando em uma das 20 cidades estudadas. Para o processamento foi utilizado o kit AmpFLSTR Identifiler Direct PCR Amplification® seguindo as fases de genotipagem, amplificação e eletroforese capilar. Foi possível estabelecer o perfil genético de 259 amostras bem como os parâmetros forenses e, assim, calcular os loci mais polimórficos os quais foram FGA e D18S51 utilizando os softwares GenAlEx 6.5, Arlequin 3.5 e R 2.5. A distribuição das frequências alélicas de cada loci analisado permitiu estabelecer a caracterização genética da população estudada. Foi possível confirmar que a população do Paraguai se encontra em equilíbrio Hardy-Weinberg, com uma diversidade genética intrapopulacional de 0.794915 +/- 0.398307 e interpopulacional determinada pelo índice de fixação (FST) de 0.01112. A partir desse estudo, foi possível determinar as frequências alélicas de 15 STRs utilizados no sistema CODIS nacapital e no Departamento Central do Paraguai bem como a caracterização genética e os parâmetros forenses da população estudada. Todos os loci estudados na população paraguaia foram considerados muito informativos e úteis para a solução de problemas relacionados com identificação humana na amostra analisada / One of the biggest challenges in Forensic Sciences is undoubtedly human identification. DNA has been responsible for a true revolution in identification techniques in the last decades from the study and identification of polymorphisms between certain molecular markers in individuals. The recommended markers for obtaining genetic profiles are loci of DNA microsatellite regions, also called Short Tandem Repeats (STR). The number of repetitions of STR markers is variable among individuals, creating polymorphism and thus making them excellent markers for human identification. The aim of this study was to establish the distribution of the allelic frequency of 16 STRs recommended in the CODIS system, in the capital and in the Central Department of Paraguay. We studied 300 saliva samples collected from NUCLEIC-CARD (TM) Collection Device System of Paraguayan individuals between 20 and 70 years-old, living in one of the 20 cities studied. For the processing, the AmpFLSTR Identifiler Direct PCR Amplification® kit was used following the phases of genotyping, amplification and capillary electrophoresis. It was possible to establish the genetic profile of 259 samples as well as the forensic parameters and thus to calculate the most polymorphic loci which were FGA and D18S51 using the software GenAlEx 6.5, Arlequin 3.5 and R 2.5. The distribution of the allelic frequencies of each analyzed loci allowed establishing the genetic characterization of the studied population. It was possible to confirm that the population of Paraguay is in Hardy-Weinberg equilibrium, with an intrapopulational genetic diversity of 0.8046 +/- 0.0120 and interpopulational determined by the fixation index (FST) of 0.01112. From this study, it was possible to determine the allelic frequencies of 15 STRs used in the CODIS system in the capital and in the Central Department of Paraguay, as well as the genetic characterization and forensic parameters of the studied population. All the loci studied in the Paraguayan population were considered veryinformative and useful for the solution of problems related to human identification in the analyzed sample

TetratricoPeptide Repeats or TPRs are a class of repeat proteins made up of - helices. Each repeat contains 34 amino acids that form a helix-turn-helix motif and is stabilised by short range interactions creating a non-globular fold. Tandem arrays of these repeats form stable superhelical structures. The modular nature of the TPR fold has allowed a series of consensus TPRs (CTPRs) to be designed where the number of repeat units has been varied. We have exploited the modular nature of CTPR proteins in order to create fibres via a bottom-up approach. Using Native Chemical Ligation (NCL) we have been able to trigger specific assembly of monomeric CTPR units to form extended fibrous structures up to microns in length (as viewed by TEM). This reaction proceeds at room temperature and neutral pH, with filaments observed within 12 hours. The equilibrium unfolding of CTPRs is prone to the population of partially folded states. Through studying the stability of a series of deletion mutants and using a Heteropolymer Ising model to analyse the unfolding data we have been able to design a CTPR with a conformational ‘switch’. This new CTPR was designed to populate a stable intermediate, with an exposed dimerisation interface, under certain conditions. When this new construct was analysed using 2D NMR and CD spectroscopy, it was found to selectively unfold its C-terminal -helix at a specific concentration of GuHCl. Our aim is to develop a system in which a ‘switching’ CTPR is used as a sensor that, when triggered by environmental conditions, partially unfolds and oligomerises.