Dissertations / Theses on the topic 'Repetitive DNA sequence'

Thesis (M.S.)--University of Missouri-Columbia, 2005.
"May 2005" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Includes bibliographical references.

Three genomic clones encompassing human DNA segments (designated LhX-3, LhX-4, and LhX5) were isolated from an X chromosome-specific library and subjected to analysis by physical mapping and DNA sequencing. It was found that these three clones are very rich in repetitive DNA sequence elements and retropseudogenes.

Microsatellites are simple repetitive DNA sequences that are used as genetic markers throughout the biological sciences. The high levels of variation observed at microsatellite loci contribute to their utility in studies at the population and individual levels. This variation is a consequence of mutations that change the length of microsatellite repeat tracts. Current understanding suggests that most mutations are caused by polymerase slippage during DNA replication and lead to changes of a single repeat unit in length, but some changes involving multiple repeats can also occur. Despite this simplistic overview, there is evidence for considerable heterogeneity in mutation processes between species, loci and alleles. Such complex patterns suggest that other mechanisms, including those associated with DNA recombination, are also involved in the generation of microsatellite mutations. Understanding which mutational mechanisms are responsible for variation at microsatellite markers is essential to enable accurate data interpretation in genotyping projects, as many commonly used statistics assume specific mutation models. I developed microsatellite markers specific to the X and Y chromosomes and an autosome in the tammar wallaby, Macropus eugenii, and investigated their evolutionary properties using two approaches: indirectly, as inferred from population data, and directly, from observation of mutation events. First, I found that allelic richness increased with repeat length and that two popular mutation models, the stepwise mutation model and the infinite allele model, were poor at predicting the number of alleles per locus, particularly when gene diversity was high. These results suggest that neither model can account for all mutations at tammar wallaby microsatellites and hint at the involvement of more complex mechanisms than replication slippage. I also determined levels of variation at each locus in two tammar wallaby populations. I found that allelic richness was highest for chromosome 2, intermediate for the X chromosome and lowest for the Y chromosome in both populations. Thus, allelic richness varied between chromosomes in the manner predicted by their relative exposure to recombination, although these results may also be explained by the relative effective population sizes of the chromosomes studied. Second, I used small-pool PCR from sperm DNA to observe de novo mutation events at three of the most polymorphic autosomal markers. To determine the reliability of my observations I developed and applied strict criteria for scoring alleles and mutations at microsatellite loci. I observed mutations at all three markers, with rate variation between loci. Single step mutations could not be distinguished because of the limitations of the approach, but 24 multi-step mutations, involving changes of up to 35 repeat units, were recorded. Many of these mutations involved changes that could not be explained by the gain or loss of whole repeat units. These results imply that a large number of mutations at tammar wallaby microsatellites are caused by mechanisms other than replication slippage and are consistent with a role for recombination in the mutation process. Taken as a whole, my results provide evidence for complex mutation processes at tammar wallaby microsatellites. I conclude that careful characterisation of microsatellite mutation properties should be conducted on a case-by-case basis to determine the most appropriate mutation models and analysis tools for each locus. In addition, my work has provided a set of chromosome-specific markers for use in macropod genetic studies, which includes the first marsupial Y chromosome microsatellites. Sex chromosome microsatellites open a new range of possibilities for population studies, as they provide opportunities to investigate gene flow in a male context, to complement data from autosomal and maternally-inherited mitochondrial markers.

Thesis (Ph. D.) -- University of Maryland, College Park, 2006.
Thesis research directed by: Applied Mathematics and Scientific Computation Program. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

A localização cromossômica do DNA ribossômico (rDNA) foi estudada em cromossomos politênicos e em tecidos diplóides de quatro espécies de sciarídeos: Trichosia pubescens; Rhynchosciara americana; R. milleri e Schwenkfeldina sp.. Resultados de hibridação em cromossomos mitóticos mostraram a existência de um único locus de rDNA; entretanto, sondas ribossomais hibridaram em mais de uma região dos cromossomos politênicos em todas as espécies analisadas devido à adesão de micronucléolos em regiões específicas dos cromossomos. Os micronucléolos são estruturas arredondadas que contêm, provavelmente, DNA extracromossômico transcricionalmente ativo. Em T. pubescens, o rDNA está predominantemente localizado nas secções cromossômicas X-10 e X-8. Em R. americana o rDNA está freqüentemente associado à heterocromatina centromérica dos cromossomos X, C, B e A, e também às secções X-1 e B-13. Sondas ribossômicas em R. milleri hibridaram, em alta freqüência, em regiões teloméricas e pericêntricas de cromossomos politênicos. Schwenkfeldina sp. apresenta uma distribuição incomum do rDNA em núcleos politênicos, caracterizada pela adesão de micronucléolos em muitas regiões cromossômicas. Os resultados mostraram que os micronucléolos estão preferencialmente associados à heterocromatina intercalar ou terminal de todos os sciarídeos analisados e, dependendo da espécie, estão aderidos a um número pequeno (Trichosia), moderado (Rhynchosciara) e grande (Schwenkfeldina sp.) de sítios em cromossomos politênicos. Este trabalho também descreve a caracterização de seqüências presentes nas extremidades cromossômicas de R. americana, que se iniciou através da triagem de uma microbiblioteca plasmidial, feita a partir de uma extremidade microdissecada B-1. Uma repetição do tipo satélite foi identificada e sua composição de bases, estrutura genômica e localização cromossômica são semelhantes às repetições teloméricas complexas de Nematocera que já foram descritas. Contudo, dados obtidos em outras espécies de Rhynchosciara, assim como a localização desse satélite e da transcriptase reversa, sugerem que o elemento repetitivo caracterizado neste trabalho não atinge as extremidades dos cromossomos. A caracterização de seqüências terminais e subterminais presentes nos cromossomos de R. americana foi continuada através da triagem de uma biblioteca de DNA desse díptero clonada em fagos Dash. Escolhemos como sonda para a triagem o clone pRaM47.33, representativo do elemento repetitivo M22, caracterizado em R. americana. Foram analisados cerca de 12kb de um único inserto de fago, que continha, alem das repetições M22, uma nova repetição de 16pb, organizada em tandem e que denominamos de M16. Resultados de hibridações in situ revelaram a presença da repetição M16 nas 5 extremidades cromossômicas não-telocêntricas de R. americana. Essa repetição também foi utilizada como sonda em uma outra triagem da mesma biblioteca genômica, o que permitiu a seleção e análise de aproximadamente 50kb de DNA cromossômico terminal de R. americana. Encontramos também, ao longo dessas 50kb de DNA analisado, repetições de 414pb anteriormente caracterizadas em R. americana; parte de seqüências do transposon Ramar1 e do retrotransposon RaTART . Além disso, foram observadas também seqüências que não apresentam semelhança significativa com seqüências depositadas no banco de dados GenBank, e que tampouco apresentam motivos repetitivos. Os resultados obtidos apontam para a possibilidade de que a região telomérica de R. americana seja composta por mais de um tipo de elemento repetitivo.
The chromosomal localization of ribosomal DNA (rDNA) was studied in polytene and diploid tissues of four sciarid species, Trichosia pubescens, Rhynchosciara americana, R. milleri and Schwenkfeldina sp. While hybridization to mitotic chromosomes showed the existence of a single rDNA locus, ribosomal probes hybridized to more than one polytene chromosome region in all the species analyzed as a result of micronucleolar attachment to specific chromosome sites. Micronucleoli are small, round bodies containing transcriptionally active, probably extrachromosomal rDNA. In T. pubescens the rDNA is predominantly localized in chromosome sections X-10 and X-8. In R. americana the rDNA is frequently found associated with centromeric heterochromatin of the chromosomes X, C, B and A, and also with sections X-1 and B-13. Ribosomal probes in R. milleri hybridized with high frequency to pericentric and telomeric regions of its polytene complement. Schwfenkfeldina sp. displays a remarkably unusual distribution of rDNA in polytene nuclei, characterized by the attachment of micronucleoli to many chromosome regions. The results showed that micronucleoli preferentially associate with intercalary or terminal heterochromatin of all sciarid flies analyzed and, depending on the species, are attached to a few (Trichosia), moderate (Rhynchosciara) or a large (Schwenkfeldina sp.) number of polytene chromosome sites. This work also describes the characterization of chromosome end sequences of Rhynchosciara americana, initiated with the screening of a plasmid microlibrary made from a microdissected polytene chromosome end. We report the identification and sequencing of an R. americana satellite displaying base composition, genomic structure and chromosomal localization similar to the complex telomeric repeats of Nematocera that have previously been characterized. However, data obtained in other Rhynchosciara species, as well as distinct chromosomal localization of satellite and reverse transcriptase loci in R. americana, suggest that the repetitive element characterized does not reach the very end of the chromosome. The characterization of chromosome end sequences of Rhynchosciara americana continued with the screening of a phage library made with its genomic DNA. We choose pRaM47.33, a clone whose insert is a repetitive microsatellite characterized in the subtelomeric region of R. americana chromosomes, as a probe for the screening. We analyzed 12kb of a single phage insert, composed of M22 tandem arrays and a new microsatellite which was 16pb long, arranged in tandem (named M16). In situ hybridization showed the presence of M16 repeats in the five telomeric termini of R. americana chromosomes. The M16 repeat was used as a probe in another screen of the same phage library, which allowed us to analyze approximately 50kb of terminal DNA. We find that repetitive sequences, such as the 414pb repeat previously characterized in R. americana and stretches of Ramar1 and RaTART mobile elements, also characterized in R. americana, compose the subtelomeric region of R. americana chromosomes. Additionally, we find sequences that do not match sequences in the GenBank database and do not present repetitive motifs. Our results suggest that the telomeric regions of R. americana chromosomes are composed of more than one type of repetitive sequence.

Cette etude concerne les regions d'adn delimitant le domaine des genes alpha globine du poulet. Dans les cellules de lignee erythroide, ce domaine est limite de part et d'autre par des regions en interaction permanente avec la matrice nucleaire. Les regions internes au domaine sont associees a la matrice pendant la transcription des genes. Les segments delimitant ce domaine se localisent a l'interieur ou a proximite de regions riches en aet t, comprenant aussi des sequences repetees. Une sequence riche en at contenant 4 sites homologues au consensus definis pour la topoisomerase ii, et un element repetifif appartenant a la famille cr1 a ete identifie dans la sequence du segment delimitant le domaine du cote 3'. Un element cr1 a l'interieur du gene epsilon globine de poulet a ete caracterise, cette structure permet d'envisager l'existence d'au moins deux mecanismes moleculaires alternatifs de dispersion de cette famille de sequence dans le genome du poulet. La sequence cr1 contient un site d'interaction pour un facteur nucleaire present dans les cellules erythroides

Knowledge of phylogenetic relationships among taxa is essential for comparative and evolutionary genomic research. Here, we report reconstruction of the phylogenetic tree of the genus Gossypium containing cultivated cottons of importance in agriculture by using variation of nuclear repetitive DNA sequences. Genomic DNA was isolated from 87 available accessions of 35 species representing all eight basic genome groups of the genus Gossypium and analyzed to infer phylogeny of the genus and genome origin of its polyploid species. Twenty-two interspersed repeated sequence clones derived from G. hirsutum, each representing a repeated sequence family, were hybridized to the genomic DNA of the 35 species, respectively. Southern hybridization showed that 15 of the repetitive DNA sequences could be detected in all of the eight diploid genome groups, five were A genome-specific, and two were detected in some of the non D-genome groups. A total of 642 major restriction bands of repeated sequences were used for phylogenetic analysis of the species. A phylogenetic tree of the species was constructed, based on the parsimony method and evaluated by the bootstrap approach. The tree was consistent with those previously constructed with different methods in major clades in which the genealogical lineages of species are largely congruent with genome designations and geographical distribution; but significantly different branching among some of the species was observed. These results not only further confirm the previously phylogenetic analysis of the species and the utility of repetitive DNA sequences for phylogenetic analysis of the genus Gossypium, but also provide new insights into the phylogeny of the genus.

The recent explosion of DNA sequence information has provided compelling evidence for the following facts. (1) Simple repetitive sequences-microsatellites and minisatellites occur commonly in the human genome and (2) these repetitive DNA sequences could play an important role in the regulation of various genetic processes including modulation of gene expression. These sequences exhibit extensive polymorphism in both length and the composition between species and between organisms of the same species and even cells of the same organism. The repetitive DNA sequences also exhibit structural polymorphism depending on the sequence composition. The functional significance of repetitive DNA is a well-established fact. The work done in many laboratories including ours has conclusively documented the functional role played by repetitive sequences in various cellular processes. Structural studies have established the sequence requirement for various non-B DNA structures and the functional significance of these unusual DNA structures is becoming increasingly clear. The structures that were characterised earlier purely from conformation point of view have aroused interest after the recent realisation that these structures could be formed in vivo when cloned in a supercoiled plasmid. The discovery of novel type of dynamic mutations where intragenic amplifications of trinucleotide repeats is associated with phenotypic changes causing many neurodegenerative disorders has provided the most compelling evidence for the importance of simple repeats in the etiology of these disorders. Secondary structures adopted by these simple repeats is a common causative factor in the mechanism of expansion of these repeats. This realisation prompted many investigations into the relationship between the DNA sequence, structure and molecular basis of dynamic mutation. Many experimental evidences have implicated paranemic DNA structures in various biological processes, especially in the regulation of gene expression. Earlier work done in our laboratory on the structure function relationship of repetitive DNA sequences provided experimental evidence for the role of paranemic DNA structure in the regulation of gene expression. It was demonstrated that intramolecular triplex potential sequences within a gene downregulate its expression in vivo (Sarkar and Brahmachari (1992) Nucleic Acids Res., 20, 5713-5718). Similarly the effect of cruciform structure forming sequences on gene expression was also documented. Sequence specific alterations in DNA structures were studied in our laboratory using a variety of biophysical and biochemical techniques. An intramolecular, antiparallel tetraplex structure was proposed for human telomeric repeat sequences (Balagurumoorthy, et al., (1994) J. Biol. Chem., 269, 21858-21869). The telomeric repeats are not only present at the end of chromosomes but they are also present at many interstitial sites in the human genome. Database search reveals that the human telomeric sequences as well as similar sequences with minor variations are present at many locations in the human genome. Telomeric repeats are GC rich sequences with the G rich strand protruding as a 3' end overhang at the end of chromosomes. When human telomeric repeats are cloned in a supercoiled plasmid, the C rich strand adopts a hairpin like conformation where as the G-rich strand extrudes into a quadruplex structure. However, the biological significance of these structures in vivo still remains to be elucidated completely. The role of a putative tetraplex DNA structure in the insulin gene linked polymorphic region of the human insulin gene in vivo in the regulation of expression of the insulin gene has been suggested. In this context, we have addressed the question whether the telomeric repeats when present within a gene affect its expression in vivol If so, what would be the possible mechanism? An attempt has been made to understand the effect of presence of telomeric repeats within a gene on its expression. The details of these studies have been presented in Chapter 2 of this thesis. Contrary to telomeric repeats which provide stability to the chromosomes, recently expansion of a GC rich dodecamer repeat upstream of cystatin B gene (chromosome 21q) has been shown to be the most common mutation associated with Progressive Myoclonus Epilepsy (EPM1) of Unverricht-Lundberg type. Two to three copies of the repeat (CCCCGCCCCGCG)n are present in normal individuals whereas the affected individuals have 30-75 copies of this repeat. The expression of cystatin B gene is reduced in patients in a cell specific manner. The repeat also shows intergenerational variability. The exact mechanism of expansion of this repeat is not known. In the case of trinucleotide repeat expansion, it is shown that the structure adopted by the repeat plays an important role in the mechanism of expansion and that some of the secondary structures adopted by trinucleotide repeats could be inherently mutagenic conformations. In order to understand the mechanism of expansion EPM1 dodecamer repeat, the work reported in this thesis was carried out with the following objectives. • To understand the structure of G rich and C-rich strands of EPM1 repeat. • To understand the variations in the structure with the increase in the length and its possible implications in the mechanism of expansion of EPM 1 repeat. Studies aimed with these objectives are presented in chapters 3, 4 and 5 of the thesis. Chapter 1 provides a general introduction to repetitive DNA, the various structures adopted by repetitive DNA sequences in the genome, the functional significance of the various simple repetitive DNA sequences in the genome has been presented. An account of trinucleotide repeat expansion and associated disorders, non-trinucleotide repeat expansions and associated disorders has been presented. The various non B-DNA structures adopted these repeats and their implications in the mechanism of expansion have been discussed. Chapter 2 describes in frame cloning of human telomeric repeats d(G3T2A)3G3 in the N-terminal region of β-galactosidase gene. The effect of such repeat Sequences on transcription elongation in vivo has been studied using E.coli as a model system. The 3.5 copies of human telomeric repeat sequences were cloned in the sense strand of plasmid pBluescriptllSK+ so as to create plasmid clone pSBQ8 and in the template strand of plasmid pBluescriptHKS+ so as to create clone pSBRQ8. One dimensional chloroquine gel shift assay indicated presence of an unwound structure in pSBQ8 and pSBRQ8. β-galactosidase activity assay suggested downregulation of the gene in vivo. In the case of plasmid pSBQ8 the difference in β-galactosidase activity was approximately 6 fold as compared to the parent plasmid pBluescriptIISK+ whereas in the case of pSBRQ8 the difference in β-galactosidase activity was approximately 8 fold as compared to the control pBluescriptIIKS+. The analysis of β-galactosidase transcript showed that full length transcript was formed in the case of pSBQ8. Full length transcript was not formed in the case of pSBRQ8. We propose that in the case of pSBQ8 the gene expression is inhibited in steps subsequent to transcription elongation. In the case of pSBRQ8, we propose that quadruplex structure may be formed by the template strand at the DNA level thereby blocking transcription elongation step. Chapter 3 describes studies aimed at understanding the structure of G-rich strand (referred to as G strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the G strand of dodecamer EPM1 repeat is d(GGGGCGGGGCGC)n. Oligoucleotides containing one (12mer), two (24mer) and three(36mer) were synthesised. These oligonucleotides are referred to as dG12, dG24 and dG36 respectively. Structural studies were carried out using CD spectroscopy, UV melting, non-denaturing gel electrophoresis and chemical and enzymatic probing. The G strand oligonucleotides showed enhanced gel elecrophoretic mobility in the presence of monovalent cations KCl and NaCl. Oligonucleotide dG12 also showed retarded species on non-denaturing gel in the presence of 70mM KCl indicating intermolecular associations. Oligonucleotides dG24 and dG36 predominantly formed intramolecular structures which migrated anomalously faster than the expected size. The CD spectrum for dG12 showed an intense positive band at 260nm and a negative band at 240nm in the presence of KCl indicative of an intermolecular, parallel G quartet structure. The CD spectra of dG24 and dG36 showed 260nm positive peak, 240nm negative peak along with a positive band around 290nm. This is indicative of folded back structure. These findings support the results of non-denaturing gel electrophoresis of G strand oligonucleotides. The UV melting profiles suggested increase in the stability with the increase in the length. These structures were further characterised by PI nuclease and chemical probing using DMS and DEPC. The structural studies with G-rich strand of EPM1 dodecamer repeat showed that this repeat motif adopts intramolecularly folded structures with increase in the length of the repeat thereby favouring slippage during replication. Chapter 4 deals with the studies aimed at understanding the structure at acidic pH of C-rich strand (referred to as C strand) of Progressive Myoclonus Epilepsy (EPM1) repeat. The sequence of the C strand of dodecamer EPM1 repeat is d(CCCCGCCCCGCG)n. The C rich oligonucleotides are known to form a four stranded structure called i-motif at acidic pH involving intercalated base pairs. The i-motif consists of two parallel stranded, base paired duplexes are arranged in an antiparallel orientation. Since, the base pairs of one base paired duplex intercalate into those of the other duplex, the structure is called as i-motif. We have investigated structure of C strand of EPM1 repeat by circular dichroism (CD), native polyacrylamide gel electrophoresis and UV melting. Oligonucleotide dC12 showed two bands of which the major band was retarded on the native gel (pH 5.0) at low temperature suggesting that dC12 predominantly formed intermolecular structure, Oligonucleotides dC24 and dC36 migrated anomalously faster than the expected size indicating formation of compact, intramolecularly folded structures. Circular dichroism studies indicate that, all the oligonucleotides displayed an intense positive band near 285nm, a negative band around 260nm with a cross over at 270nm, This is a characteristic CD signature for an i-motif structure and reflects the presence of secondary structure due to formation of hydrogen bonded pairs between protonated cytosines. All the C strand oligonucleotides showed hyperchromism at 265nm, which is an isobestic wavelength for C protonation. Studies described in this chapter suggest an intramolecular i-motif structure for dC24 and dC36 and an intermolecular i-motif for oligonucleotide dC12. In addition, it was interesting to note that inspite of the presence of G residues, the stretch of C residues could adopt i-motif structure. Although these structures are formed at an acidic pH, it is indicative of formation of possible intramolecularly folded structure. Many reports have suggested the possibility of cytosine rich sequences adopting i-motif structure even at neutral pH. In order to test this possibility, structural studies were carried out on the C strand EPM1 oligonucleotides at pH 7.2 in the presence of 70mM NaCl. These studies have been described in Chapter 5. The investigations were done using CD spectroscopy, UV melting, native polyacrylamide gel electrophoresis, and chemical probing using hydroxylamine and PI nuclease. These studies indicate that all the C strand oligonucleotides form intramolecular, hairpin structure at physiological pH. All the three C strand oligonucleotides migrated anomalously faster on the native gel indicating the presence of a compact structure. The CD spectra at pH 7.2 showed a blue shift as compared to those at pH 5.0. This indicated absence of base pairs. The hydroxylamine chemical probing suggested presence of G-C Watson-Crick base pairs. The loop residues of the folded back hairpin structures were probed with PI nuclease. The C strand oligonucleotides showed possibility of formation of multiple hairpin structures with the increase in the length of the repeat. The propensity to form hairpin structures suggests a possibility of formation of slip loop structures during the replication process thereby promoting expansion of this repeat. Formation of folded back hairpin like structures is significant in terms of mechanism of expansion of this repeat. Chapter 6 is devoted to concluding remarks highlighting the significance of the experimental results presented in this thesis and their possible biological implications in the light of contemporary research.

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Universidade Estadual Paulista (UNESP)
O genoma dos organismos eucariotos apresenta-se organizado em seqüências simples e repetidas. As seqüências repetidas de DNA estão presentes em centenas a milhares de cópias dispersas ou agrupadas no genoma e localizam-se preferencialmente em regiões heterocromáticas, desempenhando papel relevante na organização do genoma desses organismos. Nesse sentido, a realização de estudos genéticos básicos sobre a organização genômica dessas seqüências repetidas é fundamental para uma melhor compreensão do seu papel biológico assim como o entendimento de sua dinâmica evolutiva entre os diversos grupos de vertebrados. Os Cichlidae constituem uma das mais especiosas famílias de peixes, com cerca de 3.000 espécies distribuídas pela América Central e do Sul, África, e sudeste da Índia. Este grupo passou por um rápido e extenso processo de radiação adaptativa ao longo dos tempos, constituindo-se em importantes entidades biológicas para a realização de estudos evolutivos. Dentre os Cichlidae, as espécies do gênero Cichla (tucunarés), com distribuição exclusiva na América do Sul, apresentam grande importância ecológica e econômica. No entanto, estudos genéticos envolvendo espécies desse gênero são ainda escassos. Assim, o presente trabalho teve por objetivo isolar e caracterizar seqüências repetidas de DNA no genoma de Cichla kelberi. Elementos repetidos de DNA foram isolados por PCR (elementos Rex1, Rex3, Rex6 e Tc1) e digestão enzimática (elemento Tuc), seqüenciados e mapeados cromossomicamente por FISH para o estudo de seu padrão de distribuição no genoma. O elemento Tuc apresentou elevada similaridade com seqüências do gene da transcriptase reversa de Oryzias melastigma, o que sugere tratar-se de um elemento retrotransponível. Análises comparativas do elemento Tuc a bancos de sequência mostraram alta similaridade...
The genome of eucaryote organisms is organized into single and repetitive sequences. The repetitive DNA sequences are represented by hundreds to thousands of dispersed or tandem-arrayed copies preferentially localized on the heterochromatic regions, having important function on the genome organization of the organisms. Therefore, the development of basic genetic studies about the genome organization of these repetitive sequences are fundamental to a better comprehension of their biologic role and the understanding of their evolutionary dinamics. The Cichlidae are one of the most diverse fish families, having about 3.000 species distributed around Central and South America, Africa and Southeast India. This group underwent a large and rapid process of adaptative radiation, becoming an important biological model. Among the Cichlidae, the species of the genera Cichla (tucunarés), with exclusive distribution in South America, have a significative economic and ecologic importance. However genetic studies on species of this genera are scarce. Therefore, this work had the aim to isolate and characterize repetitive DNA sequences of the genome of Cichla kelberi. Repetitive DNA sequences were isolated using PCR (elements Rex1, Rex3, Rex6 and Tc1) and restriction digestion (element Tuc), sequenced and their genome distribution determined by FISH. The Tuc element showed high similarity to sequences of reverse transcriptase gene of the fish Oryzias melastigma, which suggests that such element correspond to an retrotransposon element. Comparative analysis of the Tuc element to DNA sequence data bank showed high similarity with repetitive sequences in the genome of several vertebrates, including fishes, amphibians and mammals. Results of FISH showed an accumulation of obtained elements preferentially in centromeres of all chromosomes of the complement, and few telomeric blocks in some... (Complete abstract click electronic access below)

Orientador: Cesar Martins
Banca: Luciana Bolsoni
Banca: Maeli Del Paiva
Resumo: O genoma dos organismos eucariotos apresenta-se organizado em seqüências simples e repetidas. As seqüências repetidas de DNA estão presentes em centenas a milhares de cópias dispersas ou agrupadas no genoma e localizam-se preferencialmente em regiões heterocromáticas, desempenhando papel relevante na organização do genoma desses organismos. Nesse sentido, a realização de estudos genéticos básicos sobre a organização genômica dessas seqüências repetidas é fundamental para uma melhor compreensão do seu papel biológico assim como o entendimento de sua dinâmica evolutiva entre os diversos grupos de vertebrados. Os Cichlidae constituem uma das mais especiosas famílias de peixes, com cerca de 3.000 espécies distribuídas pela América Central e do Sul, África, e sudeste da Índia. Este grupo passou por um rápido e extenso processo de radiação adaptativa ao longo dos tempos, constituindo-se em importantes entidades biológicas para a realização de estudos evolutivos. Dentre os Cichlidae, as espécies do gênero Cichla (tucunarés), com distribuição exclusiva na América do Sul, apresentam grande importância ecológica e econômica. No entanto, estudos genéticos envolvendo espécies desse gênero são ainda escassos. Assim, o presente trabalho teve por objetivo isolar e caracterizar seqüências repetidas de DNA no genoma de Cichla kelberi. Elementos repetidos de DNA foram isolados por PCR (elementos Rex1, Rex3, Rex6 e Tc1) e digestão enzimática (elemento Tuc), seqüenciados e mapeados cromossomicamente por FISH para o estudo de seu padrão de distribuição no genoma. O elemento Tuc apresentou elevada similaridade com seqüências do gene da transcriptase reversa de Oryzias melastigma, o que sugere tratar-se de um elemento retrotransponível. Análises comparativas do elemento Tuc a bancos de sequência mostraram alta similaridade... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The genome of eucaryote organisms is organized into single and repetitive sequences. The repetitive DNA sequences are represented by hundreds to thousands of dispersed or tandem-arrayed copies preferentially localized on the heterochromatic regions, having important function on the genome organization of the organisms. Therefore, the development of basic genetic studies about the genome organization of these repetitive sequences are fundamental to a better comprehension of their biologic role and the understanding of their evolutionary dinamics. The Cichlidae are one of the most diverse fish families, having about 3.000 species distributed around Central and South America, Africa and Southeast India. This group underwent a large and rapid process of adaptative radiation, becoming an important biological model. Among the Cichlidae, the species of the genera Cichla (tucunarés), with exclusive distribution in South America, have a significative economic and ecologic importance. However genetic studies on species of this genera are scarce. Therefore, this work had the aim to isolate and characterize repetitive DNA sequences of the genome of Cichla kelberi. Repetitive DNA sequences were isolated using PCR (elements Rex1, Rex3, Rex6 and Tc1) and restriction digestion (element Tuc), sequenced and their genome distribution determined by FISH. The Tuc element showed high similarity to sequences of reverse transcriptase gene of the fish Oryzias melastigma, which suggests that such element correspond to an retrotransposon element. Comparative analysis of the Tuc element to DNA sequence data bank showed high similarity with repetitive sequences in the genome of several vertebrates, including fishes, amphibians and mammals. Results of FISH showed an accumulation of obtained elements preferentially in centromeres of all chromosomes of the complement, and few telomeric blocks in some... (Complete abstract click electronic access below)
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The elucidation of the composition and organization of genomes of higher plants is a fundamental problem of modern molecular biology. The genus Beta containing 14 species assigned to the sections Beta, Corollinae, Nanae and Procumbentes provides a suitable system for the comparative study of the nuclear genomes. Sugar beet Beta vulgaris has a genome size of 758 Mbp DNA with estimated 63 % repetitive sequences and the number of chromosomes n=9. The wild beet Beta procumbens is an important natural pool of resistance against pests and tolerance to unfavorable growth conditions. The subject of this research was the isolation and description of new repetitive DNA families from genomes of this Beta species. This work presents the molecular investigation and cytogenetic characterization by high-resolution multicolor fluorescent in situ hybridization (FISH) of the satellite and dispersed repetitive sequences in wild and cultivated beet species and in their hybrids. New repetitive sequences were isolated from the B. procumbens genome. The AluI restriction satellite repeats pAp11 are 229-246 bp long and form subfamilies. The satellite is amplified in the section Procumbentes, but also found in distantly related section Beta. Thus, pAp11 is probably an ancient component of Beta genomes. It could be the ancestor of the satellite subfamily pEV4 in B. vulgaris based on sequence analysis, Southern hybridization and comparative FISH. pAp11 was found at centromeric and a few intercalary sites in B. procumbens and formed intercalary blocks on B. vulgaris chromosomes where it co-localized with pEV4. These remarkable differences in the chromosomal position of pAp11 between Procumbentes and Beta species indicate that both satellites were likely involved in the expansion or rearrangement of the intercalary heterochromatin of B. vulgaris. Other two sequence families characterized on molecular, genomic and chromosomal levels are the non-homologous repeats pAp4 and pAp22, 1354 and 582 bp long. They have a dispersed organization in the genome and are widely scattered along B. procumbens chromosomes. pAp4 and pAp22 are specific for the section Procumbentes and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. High-resolution FISH on meiotic chromosomes showed that the both sequences mostly co-localize. The PCR analysis of their flanking regions revealed that pAp22 is a part of a Long Terminal Repeat (LTR) of an Athila-like env-class retrotransposon. This is the first indication that the retrovirus-like DNA elements exist in Beta. An ancient family of subtelomeric satellite DNA pAv34 was isolated from all four sections of the genus Beta and from spinach, a related Chenopodiaceae. Five clones were analyzed from each of the five species. The genomic organization and species distribution of the satellites were studied by sequencing and Southern hybridization. The repeating units in all families are 344-362 bp long and share 46.2-98.8 % similarity. Each monomer consists of two subunits SU1 and SU2 of 165-184 bp. The maximum likelihood and neighbor joining analyses of the 25 subtelomeric satellite monomers and their subunits indicated, that the duplication leading to the emergence of the 360 bp satellite should have occurred early in the phylogeny. The two directions of diversification are the clustering of satellites in two groups of subunits SU1 and SU2 and the arrangement of satellite repeats in section-specific groups. The comparative chromosomal localization of the telomeric repeat, pAv34 and rDNA was investigated by multicolor FISH. B. vulgaris chromosome termini showed unique physical organization of telomeric repeat and the subtelomeric satellite, as studied by high-resolution FISH on extended DNA fibers. The estimated length of the telomeric array was 0.55 - 62.65 kb, the length of pAv34 was 5.0-125.25 kb, the spacer between these sequences spanned 1.0-16.60 kb. Eight various classes of repeats were used to characterize the minichromosomes of the sugar beet fragment addition lines PRO1 and PAT2 by comparative multi-color FISH. The study allowed to propose a schematic pattern of repetitive DNA organization on the PRO1 and PAT2 minichromosomes. PRO1 has an acrocentric minichromosome, while PAT2 possesses a metacentric or submetacentric chromosome fragment. The functional integrity of the fragment addition line centromeres was confirmed by an immunostaining localization of the proteins specific to the active kinetochore. The serine 10-phosphorylated histone H3 was detected in pericentromeric regions of the PRO1 chromosomes. The microtubuli attachment sites were visualized as parts of kinetochore complexes.

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O DNA pode estar organizado no genoma em sequências de cópias únicas e em sequências que se repetem várias vezes, denominadas sequências repetitivas. Estas sequências são constituídas basicamente por repetições em tandem (satélites, minissatélites e microssatélites) ou dispersas (transposons ou retrotransposons), e parecem estar envolvidas em diversos eventos celulares importantes, como por exemplo nos processos de replicação do DNA, de recombinação, de expressão gênica e de evolução dos cromossomos, auxiliando na manutenção e propagação do material genético celular. Em nível cromossômico parecem ser responsáveis por proporções significativas das variações cariotípicas observadas em diversos grupos. O gênero Ancistrus (Siluriformes, Loricariidae) apresenta atualmente 68 espécies nominais, e uma enorme quantidade de espécies ainda não identificadas. Alguns trabalhos vêm utilizando sequências repetitivas também em análises citogenéticas e moleculares para identificação de cromossomos homólogos e marcadores cariotípicos interessantes que podem auxiliar os trabalhos de identificação de espécies. Apesar de serem amplamente estudadas em diversos organismos, há ainda muito a ser entendido sobre essas sequências e sua organização no genoma. Este trabalho teve como objetivo isolar sequências repetitivas no genoma de espécies de Ancistrus, afim de encontrar possíveis marcadores para o gênero, que pudessem contribuir para o entendimento da taxonomia deste grupo, bem como auxiliar no entendimento do processo de evolução dos cromossomos sexuais dessas espécies. Dentre as sequências isoladas está um transposon da família TC1/mariner que se mostrou presente em todos os cromossomos das espécies analisadas e dois DNAs satélites que se apresentam acumulados em regiões centroméricas de alguns cromossomos. Sendo assim, este estudo resultou em dados que poderão contribuir com o entendimento da evolução cariotípica do gênero Ancistrus bem como fornecer mais informações sobre características e evolução dos cromossomos sexuais em peixes.
DNA may be organized in the genome as single copy sequences and as sequences that are repeated several times, called repetitive sequences. These sequences are basically constituted by tandem repeats (satellites, minisatellites and microsatellites) or scattered (transposons and retrotransposons), and seem to be involved in important cellular events such as, DNA replication process, recombination, gene expression and chromosome evolution, assisting in maintenance and spread the genetic material of cells. At chromosome level, seems to be significant proportions of karyotypic variations observed in several groups. The genus Ancistrus (Siluriformes, Loricariidae) has, actually, 68 nominal species and several species not identified yet. Repetitive sequences have been used in molecular cytogenetic analysis for identification of homologous chromosomes and interesting karyotypic markers that can assist in species identification works. Despite being widely studied in several organisms, there is still much to be understood about these sequences and their organization in the genome. This study aimed to isolate repetitive sequences in the genome of Ancistrusspecies in order to find possible markers for the genus, which could contribute to the understanding of the taxonomy of this group and to the understanding of the process of evolution of sex chromosomes of these species. Among the isolated sequences, there is a family of TC1/mariner transposon that showed to be present in all the chromosomes of the analyzed species, and two satellites DNAs that have accumulated in centromeric regions. Thus, this study resulted in data that could be contribute to understanding the karyotype evolution of Ancistrus genus as well as providing more information on the characteristics and evolution of sex chromosomes in fish.

Orientador: Fausto Foresti
Coorientador: José Carlos Pansonato Alves
Banca: Diogo Teruo Hashimoto
Banca: Vanessa Paes da Cruz
Resumo: No presente estudo, foram analisadas citogeneticamente cinco espécies de peixes pertencentes ao gênero Trichomycterus: T. diabolus, T. iheringi, T. zonatus, Trichomycterus cf. mimonha e Trichomycterus sp., provenientes de diferentes bacias hidrográficas brasileiras. Foram utilizadas as técnicas de citogenética clássica (coloração com Giemsa, localização das RONs pela marcação com nitrato de Prata e bandamento C) e cito-moleculares (Hibridação Fluorescente in situ - FISH com sondas de DNAr 5S e 18S e o snDNA U2). Todas as espécies apresentaram número diploide de 2n=54 cromossomos com variações em sua fórmula cariotípica. Trichomycterus diabolus, T. iheringi, T. zonatus e Trichomycterus cf. mimonha apresentaram seu cariótipo com 34m + 12sm + 8st e Trichomycterus sp. apresentou 36m + 10sm + 8st, além da ocorrência de cromossomos supranumerários. As espécies também revelaram diferenças em relação ao tamanho dos dois primeiros pares cromossômicos do cariótipo, onde Trichomycterus sp. e T. diabolus apresentaram o primeiro e segundo metacêntrico de tamanho semelhante e maiores em relação aos demais cromossomos, enquanto em T. zonatus, Trichomycterus cf. mimonha e T. iheringi apenas o primeiro metacêntrico foi considerado o maior par. O bandamento C evidenciou blocos heterocromáticos instersticiais nos pares 2, 3, 7, 8, 19 e 23 de T. iheringi e no par 18 de T. diabolus, sendo que as demais espécies apresentaram blocos centroméricos de diferentes tamanhos espalhados por quase todo o cariótipo. As RONs foram identificadas em apenas um par cromossômico e foram coincidentes com a hibridação fluorescente in situ realizada com a sonda para DNAr 18S, com exceção de T. zonatus e Trichomycterus sp. que apresentaram marcações centroméricas no primeiro par e em um pequeno metacêntrico, respectivamente. A hibridação com a sonda para DNAr 5S revelou resultados mais diversos, sendo detectado um par para...
Abstract: In the present study five species of fish from the genus Trichomycterus were analyzed cytogenetically, including T. diabolus, T. iheringi, T. zonatus, Trichomycterus cf. mimonha and Trichomycterus sp., collected at different Brazilian basins. The analyses involved classical (Giemsa conventional staining, localization of NORs for silver nitrate marking and C-banding) and molecular cytogenetic techniques (fluorescent in situ hybridization with ribosomal 5S and 18S, and U2 snDNA probes). All species showed diploid number of 2n = 54 chromosomes with variations in karyotype formula. Trichomycterus diabolus, T. iheringi, T. zonatus and Trichomycterus cf. mimonha presented their karyotype with 34m + 12sm + 8st and Trichomycterus sp. presented 36m + 10sm + 8st, besides the occurrence of supernumerary chromosomes. The species also reveals differences in relation to the size of the first two chromosome pairs of the karyotype, where Trichomycterus sp. and T. diabolus presented the first and second metacentric with similar size and larger than the other chromosomes, while in T. zonatus, Trichomycterus cf. mimonha and T. iheringi only the first metacentric was considered the largest pair. The constitutive heterochromatin showed interstitial blocks in pairs 2, 3, 7, 8, 19 and 23 in T. iheringi and the par 18 in T. diabolus and the other species presented centromeric blocks with different sizes spread throughout the greater part of the karyotype. NORs were identified in only one chromosome pair and were coincident with the fluorescent in situ hybridization using probes of 18S rDNA, except for T. zonatus and Trichomycterus sp. that showed additional centromeric signals on the first pair and other small metacentric, respectively. FISH using the probes of 5S rDNA was differentially distributed in the different species, with one chromosome pairs detected in T. diabolus, two pairs in T. iheringi, T. zonatus and three ...
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
No presente estudo, foram analisadas citogeneticamente cinco espécies de peixes pertencentes ao gênero Trichomycterus: T. diabolus, T. iheringi, T. zonatus, Trichomycterus cf. mimonha e Trichomycterus sp., provenientes de diferentes bacias hidrográficas brasileiras. Foram utilizadas as técnicas de citogenética clássica (coloração com Giemsa, localização das RONs pela marcação com nitrato de Prata e bandamento C) e cito-moleculares (Hibridação Fluorescente in situ - FISH com sondas de DNAr 5S e 18S e o snDNA U2). Todas as espécies apresentaram número diploide de 2n=54 cromossomos com variações em sua fórmula cariotípica. Trichomycterus diabolus, T. iheringi, T. zonatus e Trichomycterus cf. mimonha apresentaram seu cariótipo com 34m + 12sm + 8st e Trichomycterus sp. apresentou 36m + 10sm + 8st, além da ocorrência de cromossomos supranumerários. As espécies também revelaram diferenças em relação ao tamanho dos dois primeiros pares cromossômicos do cariótipo, onde Trichomycterus sp. e T. diabolus apresentaram o primeiro e segundo metacêntrico de tamanho semelhante e maiores em relação aos demais cromossomos, enquanto em T. zonatus, Trichomycterus cf. mimonha e T. iheringi apenas o primeiro metacêntrico foi considerado o maior par. O bandamento C evidenciou blocos heterocromáticos instersticiais nos pares 2, 3, 7, 8, 19 e 23 de T. iheringi e no par 18 de T. diabolus, sendo que as demais espécies apresentaram blocos centroméricos de diferentes tamanhos espalhados por quase todo o cariótipo. As RONs foram identificadas em apenas um par cromossômico e foram coincidentes com a hibridação fluorescente in situ realizada com a sonda para DNAr 18S, com exceção de T. zonatus e Trichomycterus sp. que apresentaram marcações centroméricas no primeiro par e em um pequeno metacêntrico, respectivamente. A hibridação com a sonda para DNAr 5S revelou resultados mais diversos, sendo detectado um par para...
In the present study five species of fish from the genus Trichomycterus were analyzed cytogenetically, including T. diabolus, T. iheringi, T. zonatus, Trichomycterus cf. mimonha and Trichomycterus sp., collected at different Brazilian basins. The analyses involved classical (Giemsa conventional staining, localization of NORs for silver nitrate marking and C-banding) and molecular cytogenetic techniques (fluorescent in situ hybridization with ribosomal 5S and 18S, and U2 snDNA probes). All species showed diploid number of 2n = 54 chromosomes with variations in karyotype formula. Trichomycterus diabolus, T. iheringi, T. zonatus and Trichomycterus cf. mimonha presented their karyotype with 34m + 12sm + 8st and Trichomycterus sp. presented 36m + 10sm + 8st, besides the occurrence of supernumerary chromosomes. The species also reveals differences in relation to the size of the first two chromosome pairs of the karyotype, where Trichomycterus sp. and T. diabolus presented the first and second metacentric with similar size and larger than the other chromosomes, while in T. zonatus, Trichomycterus cf. mimonha and T. iheringi only the first metacentric was considered the largest pair. The constitutive heterochromatin showed interstitial blocks in pairs 2, 3, 7, 8, 19 and 23 in T. iheringi and the par 18 in T. diabolus and the other species presented centromeric blocks with different sizes spread throughout the greater part of the karyotype. NORs were identified in only one chromosome pair and were coincident with the fluorescent in situ hybridization using probes of 18S rDNA, except for T. zonatus and Trichomycterus sp. that showed additional centromeric signals on the first pair and other small metacentric, respectively. FISH using the probes of 5S rDNA was differentially distributed in the different species, with one chromosome pairs detected in T. diabolus, two pairs in T. iheringi, T. zonatus and three ...

Die Verbindung von genetischen, physikalischen und zytologischen Daten ist entscheidend für die Genom- und Chromosomenanalyse. Obwohl Beta vulgaris (2n = 18) als wichtige Kulturpflanze und Untersuchungsobjekt der Grundlagenforschung eine intensiv analysierte Art darstellt, existiert bisher keine Verknüpfung zwischen Kopplungsgruppen (LG) und Chromosomen. B.-vulgaris-Chromosomen können zudem aufgrund fehlender morphologischer Unterscheidungsmerkmale bisher nicht einzeln identifiziert und klassifiziert werden. Somit sind zytogenetisch gewonnene Ergebnisse nicht ohne weiteres auf genetische Kopplungsgruppen und physikalische Karten übertragbar. Zytogenetische Methoden können zur Analyse struktureller Chromosomenveränderungen, zur Identifizierung und Lokalisierung von repetitiver DNA sowie zur Kartierung schwierig zu positionierender Marker verwendet werden. Ziel dieser Arbeit war es daher, ein FISH (Fluoreszenz-in-situ-Hybridisierung)-Verfahren zu etablieren, das die Kopplungsgruppen und Chromosomen der Zuckerrübe korreliert und die mikroskopische Identifizierung aller Chromosomenarme ermöglicht. Im Rahmen dieser Arbeit wurde ein FISH-Referenzkaryotyp der Zuckerrübe entwickelt. Durch ein Sondenset aus 18 BACs (bacterial artificial chromosome) sind alle Chromosomenarme der Zuckerrübe identifizierbar und werden mit den nördlichen und südlichen Enden der genetischen Kopplungsgruppen verknüpft. Somit ist eine einheitliche Nummerierung von Kopplungsgruppen und Chromosomen möglich. Durch die gleichzeitige Hybridisierung von chromosomenspezifischen BACs und den Satelliten-DNA-Sonden pAv34 und pBV VI beziehungsweise pEV und pBV wurden die Verteilungsmuster der Sequenzfamilien auf den Chromosomen ermittelt. Die gleichzeitige Hybridisierung aller vier repetitiven Sonden ergab ein chromosomenspezifisches Muster aus subtelomerischen, interkalaren und zentromerischen Signalen. Damit ist die Identifizierung aller B.-vulgaris-Chromosomen in einem einzelnen FISH-Experiment möglich. Zudem wurden dadurch die Chromosomen mit hohem Anteil an tandemartig angeordneten repetitiven Sequenzen identifiziert und die Chromosomenregionen lokalisiert, welche die Sequenzassemblierung behindern können. Sowohl das entwickelte BAC-Set als auch der Sondenpool aus repetitiver DNA unterscheiden die somatischen Metaphasechromosomen erstmals unabhängig von trisomen Linien. Da mit Hilfe der Satelliten-DNA-Sonden alle Chromosomen gleichzeitig markiert werden können, waren die spezifischen physikalischen Längen ermittelbar. Sie wurden mit den genetischen Längen der Kopplungsgruppen in Verbindung gebracht und deckten eine kopplungs-gruppenspezifische Rekombinationshäufigkeit zwischen 0,73 und 1,14 Mb/cM auf. Durch Hybridisierung der BACs und subtelomerischer beziehungsweise telomerischer Sonden auf Pachytänchromosomen wurde der Abstand der BACs sowie der in ihnen enthaltenen genetischen Marker zum physikalischen Chromosomenende abgeschätzt. An fünf Chromo-somenenden wurde ein deutlicher Abstand zwischen den Signalen des BACs und der terminalen Sonden festgestellt. Die zugehörigen Kopplungsgruppen sind demnach erweiterbar. Zudem wurden drei BACs mit nicht detektierbarem Abstand zum Chromosomenende durch FISH an gestreckten Chromatinfasern näher untersucht. Einer der drei BACs wurde eindeutig in unmittelbarer Nähe des Telomers nachgewiesen. Für dieses Ende (Chr 2N) ist die Wahrscheinlichkeit gering, dass die Kopplungsgruppe durch zusätzliche Marker erweitert werden kann; sie wird darum als abgeschlossen angesehen. Für die Enden Chr 4S und Chr 9S war der Abstand zwischen BAC und terminaler Sonde zu groß, um ihn durch Fiber-FISH zu ermitteln. Für sie sind weitere distal zu positionierende Marker wahrscheinlich. Weiterhin wurden bioinformatische Analysen an der verfügbaren B.-vulgaris-Genomsequenz RefBeet 1.0 durchgeführt. Scaffolds, welche die genetischen terminalen Marker enthalten, wurden bioinformatisch identifiziert und auf ihren Gehalt subtelomerischer und telomerischer Sequenzen untersucht. Vorhandene terminale Sequenzen sind ein Nachweis für eine terminale Lokalisierung der in-silico-Chromosomenabschnitte. Für drei Scaffolds mit zuvor ungeklärter Lage wurde dadurch das in-silico-Chromosom ermittelt beziehungsweise die nördliche oder südliche Position auf dem Chromosom dargestellt. Durch die Lokalisierung dieser Bereiche innerhalb der Sequenz in Bezug zum genetischen Marker und unter Berücksichtigung der Ergebnisse der Pachytän-FISH wurde die Strangorientierung von 16 Scaffolds ermittelt. Auf 14 Scaffolds wurden die Abstände der Marker zu den terminalen Sequenzen bestimmt. Der Median betrug etwa 196 kb. Für alle Kopplungsgruppenenden außer dem Norden von LG 2 und LG 4 ist das Vorhandensein weiterer distaler genetischer Marker wahrscheinlich. Satelliten-DNA ist innerhalb einer Art meist homogen, kann jedoch chromosomenspezifische Varianten ausbilden. Auf dem BAC-Marker für Chr 2N wurde durch Southern-Hybridisierung die subtelomerische Sequenzfamilie pAv34 detektiert. Von dem betreffenden BAC wurde eine Subklonbank erstellt. Durch Southern-Hybridisierung wurde der pAv34-Gehalt der Subklone analysiert. Positive Klone wurden sequenziert. Dabei wurden vier verschiedene vollständige pAv34-2N-Monomere detektiert. Im Vergleich mit pAv34-Volllängenmotiven aus der RefBeet 1.0 und dem Datensatz der nicht assemblierten Sequenzen der RefBeet 0.2 bilden die pAv34-2N-Einheiten mit pAv34-Kopien, die verschiedenen in-silico-Chromosomen und Contigs zugeordnet sind, eine Subfamilie. Aus den Sequenzen der Subklone wurden zwei Subklon-Contigs gebildet, die im in-silico-Chromosomenabschnitt von Chr 2N (Bvchr2.un.sca001) positioniert wurden. Dadurch wurden Regionen bisher unbekannter Sequenz entschlüsselt. Abweichungen zwischen den assemblierten Daten und den Subklonsequenzen deuten auf Assemblierungsfehler der Genomsequenz in repetitiven Bereichen hin. Die in dieser Arbeit erzielten Ergebnisse ermöglichen erstmalig die eindeutige Identifizierung aller B.-vulgaris-Chromosomen unabhängig vom Zellzyklusstadium und im Einklang mit genetischen Informationen. Zytogenetische sind jetzt mit molekularen Daten integrierbar und können verwendet werden, um den chromosomenspezifischen Satelliten-DNA-Gehalt aufzudecken und mögliche chromosomenspezifische Subfamilien zu identifizieren. Sie erlauben, physikalische Abstände zwischen Markern zu ermitteln und die Abdeckung von Kopplungsgruppen im terminalen Bereich zu untersuchen. Die Ergebnisse tragen dazu bei, Marker und nicht zugeordnete Contigs und Scaffolds zu kartieren, Ursachen für Lücken aufzudecken und damit die Sequenzdaten des Zuckerrübengenoms zu einer fortlaufenden, hochqualitativen Sequenz zu assemblieren. Die zytogenetischen Daten bilden zudem die Basis für zukünftige Untersuchungen struktureller Umbauten von Chromosomen, die während der Genomevolution stattfanden
The correlation of genetic, physical and cytological data is crucial for interdisciplinary genome and chromosome analyses. Beta vulgaris (2n = 18) is an important crop and an object of basic research. Although it is an intensely analysed species, its genetic linkage groups (LG) have not been assigned to chromosomes. Additionally, sugar beet chromosomes lack distinct morphological features and could therefore not be identified and classified individually. Consequently, results generated by cytogenetic methods can not be readily applied to genetic and physical maps. Cytogenetic approaches enable analysing structural chromosomal changes, identifying and localizing repetitive DNA, and mapping of markers which are difficult to place within linkage maps. Therefore, the main objective of this work has been the development of a FISH (fluorescence in situ hybridization) procedure that correlates LGs with chromosomes of sugar beet and that allows the microscopic identification of individual chromosome arms. In this work a FISH reference karyotype for sugar beet has been established. A set of 18 BACs (bacterial artificial chromosome) allows the unequivocal identification of each sugar beet chromosome and assigns them to the southern and northern ends of LGs. Hence, the chromosomes are numbered in accordance with the genetic map. The arm-specific BACs and the satellite DNA families pBV and pBV VI or pEV and pAv34 have been hybridized simultaneously to assign the distribution patterns of the highly abundant sequence families to chromosomes. Simultaneous hybridization of the four repetitive probes revealed a chromosome-specific pattern of subtelomeric, intercalary and centromeric signals. Thus, each of the sugar beet chromosomes can be identified in a single FISH experiment. Furthermore, chromosomes with a high content of repetitive DNA have been identified and chromosomal regions that may hinder the correct sequence assembly have been localized. The BAC set as well as the pooled satellite DNA probes discriminate the somatic chromosomes for the first time independently from trisomic lines. Since the chromosomes are differentially labelled with the satellite DNA probes their physical distances could be determined and correlated with genetic distances of the corresponding LGs. A LG-specific recombination frequency from 0.73 to 1.14 Mb/cM has been disclosed. BACs and subtelomeric or telomeric sequences have been hybridized simultaneously on pachytene chromosomes to estimate distances between BACs plus the markers they contain and the physical chromosome ends. Five BACs showed substantial distances to the physical chromosome ends; the corresponding LGs could thus be extended by additional markers. Furthermore, three BACs showing only minor distances to chromosome ends have been investigated in detail by fiber-FISH. One of these BACs was localized closely adjacent to the telomere. For this chromosome end (Chr 2N) it is unlikely that the LG could be extended distally by additional markers and is therefore considered to be closed. The BACs for the chromosome ends Chr 4S and Chr 9S have been too distant from the terminal probe to be bridged by fiber-FISH. For them it is likely that further markers can be placed distally. Furthermore, the B. vulgaris genomic sequence RefBeet 1.0 has been investigated. Scaffolds containing terminal genetic markers have been identified bioinformatically and analysed for the content of subtelomeric and telomeric sequences. The occurrence of terminal sequences confirms the terminal localization of in silico chromosome segments. Three scaffolds with an initially unknown position could thus be allocated to in silico chromosomes and to the northern or southern position on the chromosome. The strand orientation of 16 scaffolds has been determined based on the localization of terminal sequences in relation to the genetic marker considering the results of FISH on pachytene chromosomes. The distance between markers and terminal sequences has been determined for 14 scaffolds. The median is 196 kb. It is likely that further markers can be placed distally from all LG ends except for the north of LG 2 and LG 4. Satellite DNA is usually homogenous within one species; however, it can form chromosome-specific variants. Southern hybridization revealed that the BAC marker for Chr 2N contains the subtelomeric sequence family pAv34. The BAC has been subcloned and the pAv34 content of the subclones has been analysed by Southern hybridization. Positive clones have been sequenced. Thereby, four pAv34-2N monomeres have been detected. Compared to full-length pAv34 motives derived from the RefBeet 1.0 and from unassembled sequence data of the RefBeet 0.2 the pAv34-2N units form a subfamily together with pAv34 copies assigned to different in silico chromosomes and contigs. The subclone sequences have been assembled to two subclone contigs, which have been positioned within the in silico chromosome segment of Chr 2N (Bvchr2.un.sca001). Thereby, regions of unknown sequence have been decoded and probable misassemblies in repetitive regions within the RefBeet 1.0 have been disclosed. The results obtained in this work enable the identification of all sugar beet chromosomes independently from their stage of cell division and in accordance with genetic information. Cytogenetic data are integrated with molecular data and can be used for identifying the chromosome-specific distribution of repeats and chromosome-specific repeat variants. They enable determining physical distances between markers and investigating the terminal coverage of LGs. The results support the correct mapping of markers and unassigned contigs, uncover reasons for gaps within maps and sequence assemblies, and thus contribute to assembling data into a continuous high quality genome sequence of sugar beet. Moreover, the cytogenetic data represent the basis for future investigations of structural chromosomal changes that took place during evolution

Megasatellites are polymorphic tandem repetitive sequences with repeat-units longer than or equal to 1000 base pairs. The novel algorithm Megasatfinder predicts megasatellites in the human genome. A structured method of analysing the algorithm is developed and conducted. The analysis method consists of six test scenarios. Scripts are created, which execute the algorithm using various parameter settings. Three nucleotide sequences are applied; a real sequence extracted from the human genome and two random sequences, generated using different base probabilities. Usability and accuracy are investigated, providing the user with confidence in the algorithm and its output. The results indicate that Megasatfinder is an excellent tool for the detection of megasatellites and that the generated results are highly reliable. The results of the complete analysis suggest alterations in the default parameter settings, presented as user guidelines, and state that artificially generated sequences are not applicable as models for real DNA in computational simulations.

碩士
國立臺灣大學
植物學系研究所
86
Nicotiana plumbaginifolia has 10 pairs of acrocentric or telocentricchromosome s, which could not be distinguished from each other by conventional cytologica l techniques. The somatic karyotype of this species was constructed based on f luorescence in situ hybridization (FISH) to a 165 bp tandemly repeated sequenc e, NP3R. Variation in the spatial and quantitative distribution of this repeat allowed the identification of each of the 10 pairs of N. plumbaginifolia chr omosomes. Two-step FISH and multi-color FISH, showed that 5S rDNA was clustere d in a proximal region in the long arm of chromosome 2, and that 26S rDNA was clustered in the terminal regions of chromosomes 9 and 10.

碩士
國立陽明大學
生化暨分子生物研究所
97
1.Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder and is caused by the expansion of CAG repeats, encoding polyglutamines (polyQ), within exon 1 of the huntingtin (htt) gene. The length of polyQ track is a main determinant for the onset of HD and the disease occurs only when the critical length of ~36 glutamines is exceeded. A peculiar feature of mutant htt proteins with abnormal length of polyQ is that they spontaneously self-assemble into aggregates, a pathological hallmark of this disorder found in the brain of affected individuals. Several studies have demonstrated that proteins with normal length of polyQ track can also interact with and recruit into these aggregates, which are initiated by polyQ-expanded proteins. In order to further investigate this inter-molecular interaction between heterogeneous polyQ strands and to find a potential dominant negative mutant that can interfere with the formation of polyQ aggregates in cells, we have established a phenotypic assay system that can reveal the presence of co-aggregation and costructed a library of amino acid substitution within the polyQ track for examining their effect on polyQ aggregation. After three rounds of screening, we find that co-expression of truncated polyQ peptides can partially reduce the aggregation levels of 97Q protein. However, this effect may result from decreased 97Q expression. 2.The BEM2 gene is required for normal polarized cell growth in the budding yeast Saccharomyces cerevisiae. In the carboxyl-terminus, Bem2p contains a GAP domain, which is commoly found in GTPase activating proteins (GAPs) that regulate rho-GTPase activity. In yeast, genes encoding GTPases (CDC42, RHO1, RHO2, RHO3, and RHO4) have been proposed to regulate cellular morphogenesis such as actin cytoskeleton and bud emergence. Using the Snyder’s knock out library, we find that bem2△ mutations confer DNA repetitive sequence instability and enhance contraction of CAA/CAG repeats through homologous recombination, suggesting Bem2p has a role in regulating DNA stability. We also demonstrate the DNA instability is attributed to the defective function of Bem2p in morphogenesis checkpoint. Furthermore, swe1, a△ morphogenesis checkpoint deficient cell, shows an elevated DNA contraction rate. Thus, our study reveals a novel link between morphogenesis checkpoint and repetitive sequence stability in yeast cells.

碩士
國立陽明大學
生化暨分子生物研究所
102
RNA:DNA hybrids, known as R-loops, are peculiar nucleic acid structures that are generated under transcriptional process; in particular, for these genes with highly repetitive or GC rich sequences. R-loops can be dynamically resolved by a variety of cellular proteins and the persistence of RNA:DNA hybrids has been proposed as a negative factor to influence genome integrity. Recently, R-loops were detected on CAG trinucleotide repeats (TNR) and contributed DNA instability as a consequence of contraction of repetitive sequences. Here, using yeast cells as a model system, we analyzed the integrity of repetitive sequences and found CAG repeat instability is promoted by transcription per se and is dependent on the length of repetitive sequence. This DNA instability is ameliorated by ectopic expression of multiple proteins that bear the enzymatic activity to resolve R-loops. Additionally, we discovered the extent of DNA instability is influenced by the composition of nucleotide sequences. Furthermore, we found a differential effect of transcription elongation factors on the DNA instability of CAG repeats. Our collected results demonstrated the causal effect of R-loops on TNR instability, and also revealed the contribution of transcription elongation factors and TNR nucleotide sequences to this transcription-induced DNA instability.

碩士
國立中興大學
畜牧研究所
80
DNA 重複序列遍存於真核生物基因組中，已有研究者以重複序列DNA 為探針，應用 於DNA 指紋分析。本研究之目的在於探討乳牛基因組DNA 重複序列之性質，並探討 其應用於DNA 指紋法分析之可行性。同時也以商品化之人33.15 探針與荷蘭牛、豬 、雞及小鼠之基因組DNA 雜交，進行DNA 指紋分析，供對照之用。 試驗分三部分進行。第一部分，取荷蘭牛基因組DNA ，以Pst Ⅰ切割，經膠體電泳 後，在膠片上形成７條明顯的DNA環帶，並回收其第６條環帶(0.7kb) DNA，架構入 pUC18 內，利用X-gal 及碎裂法快速偵測出重組質體，然後進行DNA 序列分析，再 將之與限制切割之荷蘭牛、豬、雞及小鼠之基因組DNA 進行初步雜交試驗，以探 討其重複序列及其應用於DNA 指紋法分析之可行性。第二部分，取本研究室篩選到 並已接在pUC18 內之牛、豬、雞及小鼠之基因組DNA 片段進行初步雜交試驗，若有 多態性產生，則再進一步分析其DNA 重複序列及其應用於DNA 指紋法分析之可行性 。本試驗也應用人33.15 探針，與經限制切割、電泳及轉漬之荷蘭牛、豬、雞及 小鼠之基因組DNA 雜交，進行DNA 指紋分析法，並將牛α s1-酪蛋白基因之DNA 片 段與之作點雜交，以探討牛α s1-酪蛋白基因之DNA 片段是否與人33.15 探針具有 同源性關係。 試驗結果顯示，牛基因組DNA經PstⅠ處理，回收之第６條環帶之重組質體DNA 探針 ，與經不同濃度之PstⅠ限制切割之荷蘭牛基因組DNA雜交，在X-光片上呈現不同 濃度之梯狀分佈之雜交環帶，顯示該部分之DNA 是由聚合態(polymer) 分解成單一 態(monomer) ，且散佈在基因組中。經分析其DNA 序列，發現其為A+T-rich且其重 複序列大都屬於短核酸序列形態存在。在牛αs1-酪蛋白DNA重複序列之研究中， 發現5'端之0.9 kb之DNA 片段序列中，有４段重複序列，每段長為74-103 bp ，其 氮鹼基組成G+C 佔大部分。利用人33.15 探針與荷蘭牛、豬、雞及小鼠等經限制 切割後之基因組DNA 雜交，結果，發現部份有多態性出現。以人33.15 探針與牛α s1-酪蛋白基因17個分殖DNA片段雜交的結果，發現其中有６個極強之訊息，顯示其 中部份序列與人33.15 探針DNA 序列具有一致之相似性。

碩士
國立臺灣大學
植物學研究所
89
Two clones containing repetitive DNA sequences, pvr1-96 and pvr1-57, were isolated from Phalaenopsis violacea and characterized. The insert of pvr1-96 (3838 bp) consists of (ATAAGTC)432 central core flanking by 636 bp and 171 bp nonrepetitive sequence at the 5' and 3' ends, respectively. The insert of pvr1-57 is 4175 bp long which shows high homology to part of the polyprotein (pol) gene of Ty3/gypsy type retrotransposon from other species. Using a 205-bp fragment in the 5' region of pvr1-57 as a probe, a clone pvr283 was isolated. Combining the insert sequences of pvr283 and pvr1-57 gives a 7571-bp sequence, named PVRE1. PVRE1 shares 52 % identity to the pol gene of the Ty3/gypsy type retrotransposon from Oryza sativa. However, several stop codons are present in the major open reading frame (ORF) of PVRE1 and there are no long terminal repeats (LTRs) flanking this sequence, suggesting that PVRE1 is inactive. Southern analysis revealed that PVRE1 is present in all eight Phalaenopsis species studied. Chromosome localization by fluorescence in situ hybridization revealed that PVRE1 is dispersed throughout the genome of P. aphrodite, P. lueddemanniana, P. venosa and P. violacea, with signals being stronger in AT-rich heterochromatic region of the large chromosomes of the later two species. PVRE1 is the first retrotransposon isolated from Phalaenopsis species.

碩士
國立臺灣大學
植物學研究所
87
Two repetitive DNA sequences, PvrⅠand PvrⅡ, were isolated from Phalaenopsis violacea and characterized. The PvrⅠfamily consists of AT-rich regions at 5'' and 3'' ends and 7 bp direct subrepeats (GTAAGCC)13-22. Southern analysis and fluorescence in situ hybridization (FISH) revealed that PvrⅠ is present in P. mannii, P. amboinensis, P. venosa, P. violacea and Doritis pulcherrima and mostly located in the AT-rich subtelomeric heterochromatin of large chromosomes. The PvrⅡ family is present in all eight Phalaenopsis species studied. FISH using one clone (Pvr-95) as a probe showed that PvrⅡis dispersed on all chromosomes of P. venosa.

碩士
國立臺灣大學
植物學研究所
89
Heterochromatic knobs are enlarged and deeply stained regions in prophase chromosomes of maize. The main genetic effects of knobs are (1) neocentromere activity and (2) chromatin elimination. Two classes of tandemly repeated sequences associated with knobs have been isolated from the maize genome, the 180-bp repeat and TR-1 element (350-bp). Two segments (12 bp and 31 bp) in these repeats show homology of more than 60%, and the 180-bp repeat has a 72% similarity to a 90-bp stretch of the maize B chromosome centromere sequence. It has been suggested that the 180-bp repeat may be responsible for neocentromere activity. The 180-bp repeat has been extensively studied in maize and its relatives, but the molecular structure of the TR-1 element is not clear. In this study, we isolated TR-1 elements from various lines of maize and its relatives and analysed their structures. Our results demonstrates that TR-1 element is composed of A, B/B’, C repeats, and the unit length is 180 bp but not 350 bp as previously reported. There are some homology between segments of B/B’ and the 180-bp repeat, so we presume that 180-bp repeat is the ancestor of B/B’. The following reasons suggest that C+A may be actually a class of repetitive sequence with a unit length of 180 bp: (1) A and C show high homology in nucleotide sequences; (2) C is always before A in the clones analysed; (3) A and C have the same percentage (approximately 70%) of A+T. The role of TR-1 in neocentromere activity and the mechanism of the formation of TR-1 are discussed.

博士
國立成功大學
基礎醫學研究所
98
Human genome contains more than 50% proportion of repetitive DNA sequences, mostly involved chromosome structural diversity. The objective of this dissertation is to study of the impact of genomic repetitive sequences and DNA methylation on gene expression. Low copy repeat (LCR) is a new class of repetitive DNA element and has been implicated in many human disorders through non-allelic homologous recombination mechanism. In order to dissect and refine region-specific LCRs, we downloaded and analyzed the publicly available human genome sequences using computational approaches. We further took the common deletion region in chromosome 22q11.2 as an example to illustrate the use of real-time quantitative PCR assay to detect micro deletion in this region. The assay was validated in 122 patients with suspected DiGeorge/velocardiofacial (DGS/VCFS) syndromes. Our results indicated that the developed assay is reliable for clinical diagnosis. In addition, global hypomethylation of repetitive genomic sequences such as long interspersed nuclear element 1 (LINE-1), SINE-Alu and alpha-satellite (?-satellite) sequences has been associated with chromosomal instability in cancers. In addition, it has been reported that Epstein-Barr virus (EBV) promotes genomic instability in Burkitt's lymphoma and nasopharyngeal carcinoma (NPC). Thus, we investigated the methylation status of high repetitive genomic sequences under the expressions of different EBV proteins, and the impact on gene expression. EBV alternates between latency and lytic replication, and the lytic cycle is initiated when epigenetic silencing is reversed by Zta protein. We first examined methylation levels in a Zta-expression model to determine the genomic methylation of NPC cells upon EBV reactivation. Our data suggest that alteration of NPC cellular gene expression following EBV reactivation reflects methylation-independent Zta-mediated gene activation and not epigenetic modification of the host genome. Next, we analyzed the whole genome methylation profile in EBV-encoded oncoprotein latent membrane protein (LMP1)-expressing BJAB cells. By using methylation-sensitive NotI restriction enzyme, we studied promoter methylation pattern of individual genes at genomic scale. Although the overall methylation content was not changed dramatically, it reshuffled methylation marks of individual genes and specific regions including overmethylation of LINE-1, hypomethylation of ?-satellite, and unbalanced X chromosome inactivation. Overall, these data consist with the result that LMP1 increases DNMT3b expression. Furthermore, our data demonstrated the genes where expression level was significantly altered in BJAB-LMP1 cells are highly associated with cell cycle regulation and the biological markers of lymphoma. Finally, our data suggested that alteration of gene expression observed in BJAB-LMP1 cells is not through the change in promoter methylation. Rather, LMP1-mediated signaling may play significant role in eliciting host gene expression during EBV infection.