Hamburg, Univ., Diss., 2004. / Computerdatei im Fernzugriff.

Hamburg, University, Diss., 2004.

Retrolyc1 é um retrotransposon isolado de uma biblioteca genômica de L. peruvianum (Solanaceae). Análise da região promotora U3 deste elemento indicou a presença de duas subfamílias Retrolyc1A e Retrolyc1B que diferem entre si principalmente pela presença ou ausência de motivos repetidos (Araujo et al., 2001). Este trabalho teve como objetivo geral demonstrar a atividade transcricional de Retrolyc1 A e Retrolyc1B. Os resultados obtidos demonstram que região promotora de Retrolyc1B é um promotor funcional. Sendo que os perfis de expressão de Retrolyc1A e Retrolyc1B são distintos quando induzidos por moléculas mediadoras de resposta a defesa vegetal. Diferenças no perfil de expressão de Retrolyc1A e Retrolyc1B também são encontradas durante o tratamento auxinas naturais e sintéticas. A expressão de Retrolyc1 e do retrotransposon similar à Retrolyc1, Tnt1, é modulada ao longo do ciclo celular. Analises apontam aumento da expressão destes elementos nas fases G1/S. Já o estudo da expressão de Retrolyc1A em mutantes hormonais nas vias de auxina e citocinina reforçam a modulação positiva por auxina e sugerem uma modulação negativa por citocinina. Os resultados descritos sugerem a via de ativação de Retrolyc1A difere de Retrolyc1B. Sendo que Retrolyc1A possui sua expressão modulada durante o desenvolvimento vegetal, em particular nas células que estão em fase de divisão celular ativa assim como nos tecidos onde a auxina endógena é fundamental para a organogênese correta (raízes e frutos). / Retrolyc1 is a retroelemento originally isolated from genomic library of L. peruvianum (Solanaceae). Analysis based on promoter region of this element indicated that Retrolyc1 are composed of two subfamilies Retrolyc1A and Retrolyc1B (Araujo et al., 2001). This study aimed to demonstrate the transcriptional activity of Retrolyc1 A and Retrolyc1B. The results showed the promoter region of Retrolyc1B is functional. In addition, the expression pattern of Retrolyc1A and Retrolyc1B are different when they were induced by signaling molecules of plant defense response. The activity of promoter region of these elements is also activated during the treatment with natural and synthetic auxin. The expression analysis of Retrolyc1 and Tnt1 showed modulation along the cell cycle. Both elements have transcription level increased in phases G1 / S. In order to analyze the modulation of Retrolyc1A by hormone, its transcription level was analyzed in tomato mutants. The lower expression of Retrolyc1A in roots of mutants with defective perception in auxin pathway (dgt) and higher in defective cytokinin pathway (brt), reinforce the positive modulation by auxin and suggest a negative modulation by cytokinin. Our results suggest the route of activation of Retrolyc1A differs from Retrolyc1B. The expression of Retrolyc1A has been modulated during plant development, especially in cells in the process of cell division and active in the tissues where the endogenous auxin is essential for the correct organogenesis (roots and fruits).

Retrolyc1 é um retrotransposon isolado de uma biblioteca genômica de L. peruvianum (Solanaceae). Análise da região promotora U3 deste elemento indicou a presença de duas subfamílias Retrolyc1A e Retrolyc1B que diferem entre si principalmente pela presença ou ausência de motivos repetidos (Araujo et al., 2001). Este trabalho teve como objetivo geral demonstrar a atividade transcricional de Retrolyc1 A e Retrolyc1B. Os resultados obtidos demonstram que região promotora de Retrolyc1B é um promotor funcional. Sendo que os perfis de expressão de Retrolyc1A e Retrolyc1B são distintos quando induzidos por moléculas mediadoras de resposta a defesa vegetal. Diferenças no perfil de expressão de Retrolyc1A e Retrolyc1B também são encontradas durante o tratamento auxinas naturais e sintéticas. A expressão de Retrolyc1 e do retrotransposon similar à Retrolyc1, Tnt1, é modulada ao longo do ciclo celular. Analises apontam aumento da expressão destes elementos nas fases G1/S. Já o estudo da expressão de Retrolyc1A em mutantes hormonais nas vias de auxina e citocinina reforçam a modulação positiva por auxina e sugerem uma modulação negativa por citocinina. Os resultados descritos sugerem a via de ativação de Retrolyc1A difere de Retrolyc1B. Sendo que Retrolyc1A possui sua expressão modulada durante o desenvolvimento vegetal, em particular nas células que estão em fase de divisão celular ativa assim como nos tecidos onde a auxina endógena é fundamental para a organogênese correta (raízes e frutos). / Retrolyc1 is a retroelemento originally isolated from genomic library of L. peruvianum (Solanaceae). Analysis based on promoter region of this element indicated that Retrolyc1 are composed of two subfamilies Retrolyc1A and Retrolyc1B (Araujo et al., 2001). This study aimed to demonstrate the transcriptional activity of Retrolyc1 A and Retrolyc1B. The results showed the promoter region of Retrolyc1B is functional. In addition, the expression pattern of Retrolyc1A and Retrolyc1B are different when they were induced by signaling molecules of plant defense response. The activity of promoter region of these elements is also activated during the treatment with natural and synthetic auxin. The expression analysis of Retrolyc1 and Tnt1 showed modulation along the cell cycle. Both elements have transcription level increased in phases G1 / S. In order to analyze the modulation of Retrolyc1A by hormone, its transcription level was analyzed in tomato mutants. The lower expression of Retrolyc1A in roots of mutants with defective perception in auxin pathway (dgt) and higher in defective cytokinin pathway (brt), reinforce the positive modulation by auxin and suggest a negative modulation by cytokinin. Our results suggest the route of activation of Retrolyc1A differs from Retrolyc1B. The expression of Retrolyc1A has been modulated during plant development, especially in cells in the process of cell division and active in the tissues where the endogenous auxin is essential for the correct organogenesis (roots and fruits).

No description available.

No description available.

Long Interspersed Element 1(LINE1 or L1), along with the parasitic Short Interspersed Element (SINE), are the only two currently active retrotransposons in the human genome. These genetic elements are capable of causing DNA damage through their mobilization and the enzymatic activities of the L1-encoded Open Reading Frame 2 (ORF2) protein (ORF2p). The L1 ORF2p contains four annotated domains important to retrotransposition. These include the endonuclease (EN) and reverse transcriptase (RT) domains, as well as the Z domain and the cysteine rich domain (Cys). While much is known about the enzymatic activities of the EN and RT domains, and individual amino acids important to retrotransposition have been identified in the Z and Cys domains, more than 50% of the 150kDa ORF2p amino acid sequence serves no known function. I hypothesized that the unannotated areas of the ORF2p, specifically the sequence C-terminal to the EN domain and N-terminal to the Z domain as well as the sequence C-terminal to the Cys domain, contained amino acids important to the retrotransposition process. Specifically, I hypothesized that they contained amino acids involved in the activity of the EN domain, RT domain, or interaction with the L1 ORF1p. To test this hypothesis, I developed a technique termed Bipartile Alu Retrotransposition (BAR) that utilized EN and RT-containing ORF2 fragments combined with the Alu retrotransposition reporter construct. This system allowed me to define a new ORF2p region, which I termed Cryptic. This region contains an essential WD pair important for cDNA syntheses by the ORF2p. This WD pair is also involved in the regulation of the EN domain activity. I also discovered a putative PCNA binding domain that is essential for retrotransposition. Additionally, I identified the region in Cryptic that is involved in the previously reported differences in subcellular localization and cytotoxic potential of EN-containing ORF2p fragments. Using truncated ORF2p fragments generated for use in BAR, I also made several discoveries concerning the extreme C-terminus of the ORF2p. Notably, I discovered that the extreme C-terminal end of the ORF2p is dispensable for retrotransposition. I also identified a human-specific Y residue that is important for Alu retrotransposition driven by the ORF2p. / 1 / Claiborne Magnant Christian

Frankfurt (Main), Universiẗat, Diss., 2007.

Mobile group II introns are retroelements that are found in prokaryotes, archaea, and the organelles of plants and fungi, but not in the nuclear genomes of eukaryotes. They consist of a catalytically active RNA and intron-encoded reverse transcriptase, which together promote site-specific integration into DNA sites in a mechanism called retrohoming. The group II intron Ll.LtrB has been developed into a programmable, DNA-targeting agent called "targetron", which is widely used in bacteria and an attractive technology for gene targeting in eukaryotes. However, group II intron genome targeting in human cells has not been equivocally shown. This dissertation focuses on the hypothesis that the low Mg2+-concentrations found in higher eukaryotes present a natural barrier to group II introns. First, I studied E. coli host proteins that aid group II intron retrohoming and found that synthesis of a second DNA-strand relies on host replication restart proteins. Next, I demonstrated that mutations in the distal stem of the catalytic core domain V (DV) improve Ll.LtrB retrohoming in a low Mg2+-concentration E. coli mutant and in biochemical assays. These results suggest that DV is involved in an RNA-folding step that becomes rate limiting at low Mg2+. Subsequently, I performed directed evolution of the intron RNA by injecting in vitro prepared mutant intron libraries into Xenopus laevis oocyte nuclei. The mutations were analyzed using Roche 454 sequencing to generate an intron fitness landscape, which revealed conserved positions and potentially beneficial mutations, enabling enhanced retrohoming in Xenopus oocytes. Finally, I used a hybrid Pol II/T7 Ll.LtrB eukaryotic expression system to show that high exogenous MgCl2 in the growth media enables retrohoming into plasmids and genomic DNA in human cells. In vivo directed evolution and mutation analyses using PacBio RS circular consensus sequencing indicated that only a few mutations may improve intron activity in human cells. This dissertation provides evidence that efficient group II intron retrohoming in human cells is limited by low Mg2+-concentrations and develops new approaches for overcoming this limitation to enable use of group II introns for gene targeting in higher organisms. / text

Dissertação (mestrado)—Universidade de Brasília, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2016. / Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2016-12-05T12:10:51Z No. of bitstreams: 1 2016_ElizaFabriciodeMeloBellarddoNascimento.pdf: 1510859 bytes, checksum: f46e5594f50b95ae5eda3cd325437b0e (MD5) / Approved for entry into archive by Raquel Viana(raquelviana@bce.unb.br) on 2017-01-13T17:15:16Z (GMT) No. of bitstreams: 1 2016_ElizaFabriciodeMeloBellarddoNascimento.pdf: 1510859 bytes, checksum: f46e5594f50b95ae5eda3cd325437b0e (MD5) / Made available in DSpace on 2017-01-13T17:15:16Z (GMT). No. of bitstreams: 1 2016_ElizaFabriciodeMeloBellarddoNascimento.pdf: 1510859 bytes, checksum: f46e5594f50b95ae5eda3cd325437b0e (MD5) / O amendoim (Arachis hypogaea L.) é uma espécie alotetraplóide recente com dois subgenomas oriundos da hibridação entre as espécies silvestres diplóides, A. duranensis (genoma A) e A. ipaënsis (genoma B), seguida por uma duplicação cromossômica espontânea (3.500 a 9.400 anos atrás). O genoma de A. hypogaea é estimado em 2,8 Gb e composto em sua maioria por sequências repetitivas. Com o objetivo de compreender alterações no genoma que se seguiram após a poliploidização dos cromossomos de A. hypogaea, um híbrido alotetraploide sintético (A. ipaënsis x A. duranensis)4x, e as espécies genitoras diploides foram citogeneticamente comparados. Os cariótipos dos alotetraploides partilham muitas semelhanças derivadas de seus genitores diploides: o número e a morfologia geral dos cromossomos; a intensidade e posição de bandas heterocromáticas DAPI+ em regiões centroméricas; número de sítios de DNAr 5S; e a distribuição geral de DNA repetitivo. No entanto, um dos sítios de DNAr 5S no alotetraploide sintético mudou de posição em relação aos seus genitores. Embora para A. hypogaea, os sítios de DNAr 45S foram equivalentes à soma do encontrado nas espécies diploides, no alotetraploide sintético dois sítios foram perdidos. De maneira geral, após GISH, os cromossomos de ambos os alotetraploides mostraram hibridização preferencial com seus genomas diploides correspondentes. No entanto, pelo menos um par de cromossomos do alotetraploide sintético apresentou padrões de hibridização em mosaico, como indicativos de recombinação entre os subgenomas. Este estudo fornece provas claras de que o genoma do alotetraploide sintético é mais instável do que o genoma de A. hypogaea. Fato considerado inesperado, já que derivam das mesmas espécies genitoras. Por alguma razão, a estrutura dos cromossomos de A. hypogaea é inerentemente mais estável, ou tem sido, pelo menos parcialmente estabilizado através de alterações genéticas e seleção. ________________________________________________________________________________________________ ABSTRACT / Peanut (Arachis hypogaea L.) is a recent allotetraploid with two subgenomes derived from hybridization between the diploid wild species, A. duranensis (A genome) and A. ipaënsis (B genome), followed by spontaneous chromosomal duplication (3,500 to 9,400 years ago). The genome of A. hypogaea has a high repetitive content and estimated size of 2.8 Gb. With the aim of understanding genome changes that followed polyploidy, the chromosomes of A. hypogaea, an induced allotetraploid hybrid (A. ipaënsis x A. duranensis)4x, and the diploid progenitor species were cytogenetically compared. The karyotypes of the allotetraploids share many similarities derived from the progenitor diploids: the number and general morphology of chromosomes; the intensity and position of DAPI+ heterochromatic bands in centromeric regions; 5S rDNA loci number; and overall distribution of repetitive DNA. However, one of the 5S rDNA loci in the induced allotetraploid had changed position relative to its progenitors. Whilst for A. hypogaea, the 45S rDNA loci were equivalent to the sum of those present in the diploid species, in the induced allotetraploid, two loci have been lost. Generally, the chromosomes of both tetraploids showed preferential hybridization to their corresponding diploid genomes. However, at least one pair of chromosomes of the induced allotetraploid had mosaic hybridization patterns, indicative of recombination between subgenomes. This study provides clear evidence that the genome of the induced allotetraploid is more unstable than the genome of A. hypogaea. This could be considered unexpected because they derive from the same progenitor species. For some reason the chromosome structure of A. hypogaea is inherently more stable, or, it has been at least partially stabilized through genetic changes and selection. Key words: peanut; genome content; heterochromatic banding; rDNA; LTR-retrotransposons, GISH; FISH; chromosomes.