Orientador: Maria Luiza Silveira Mello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T20:32:58Z (GMT). No. of bitstreams: 1 Mampumbu_AndreRoberto_D.pdf: 659647 bytes, checksum: 40d0a48fa1d02750dffb30ae80b25445 (MD5) Previous issue date: 2006 / Resumo: O gênero Melipona (abelhas sem ferrão) tem sido dividido em dois grupos, com base no seu conteúdo em heterocromatina revelada com a técnica de banda-C em cromossomos mitóticos. Melipona quadrifasciata e Melipona rufiventris apresentam, respectivamente, níveis baixos e altos de heterocromatina. Na suposição de que cromatina condensada possa ser rica em seqüências de DNA metiladas, M. quadrifasciata e M. rufiventris poderiam então apresentar diferenças em conteúdo de seqüências CpG metiladas. Se isso acontecesse, as diferenças poderiam ser reveladas pela comparação de valores Feulgen-DNA obtidos por análise de imagem de células tratadas com as enzimas de restrição Msp I e Hpa II, que distinguem entre seqüências metiladas e não metiladas. Msp I e Hpa II clivam as seqüências ¿CCGG-, porém não há clivagem pela Hpa II se a citosina do dinucleotídeo central CG for metilada. Neste trabalho, túbulos de Malpighi de larva de último estádio de M. quadrifasciata e M. rufiventris submetidos à reação de Feulgen precedida pelo tratamento com Msp I e Hpa II tiveram suas células analisadas por microespectrofotometria de varredura automática. Para esse material houve necessidade do desenvolvimento prévio de um ajuste metodológico que tornasse a reação de Feulgen reveladora apenas de DNA, visto que ocorria reação plasmal; isto foi conseguido com um tratamento por boridreto de sódio a 5% e acetona/clorofórmio (1:1, v/v) antecedendo a reação de Feulgen. Também, embora a definição de altos e baixos conteúdos de heterocromatina em Melipona pela técnica de banda-C não fosse extensível à cromatina de núcleos interfásicos dos túbulos de Malpighi dessas abelhas, demonstrou-se que a depurinação do DNA em M. quadrifasciata era mais rápida do que a de M. rufiventris, confirmando, maiores teores de cromatina condensada em M. rufiventris. Os valores Feulgen-DNA para a heterocromatina de Melipona rufiventris e para a pouca heterocromatina somada a alguns domínios de eucromatina de Melipona quadrifasciata diminuíram após tratamento com Msp I, porém ficaram inalterados após tratamento com Hpa II. Conclui-se que seqüências CpG metiladas podem estar contidas em diferentes compartimentos cromatínicos, conforme a espécie do gênero Melipona considerada, e que os seus efeitos silenciadores possam atuar induzindo uma mesma fisiologia celular / Abstract: The genus Mellipona has been divided into two groups based on its heterochromatin content revealed by C-banding pattern in mitotic chromosomes. Melipona quadrifasciata and Melipona rufiventris show low and high heterochromatin content, respectively. Supposing that condensed chromatin may be rich in DNA methylated sequences, M quadrifasciata and M. rufiventris could, thus, show differences regarding their content of CpG methylated sequences. In this situation, such differences could be revealed by comparing the Feulgen-DNA values acquired after image analysis of cells treated with restriction enzymes Msp I and Hpa II, which distinguish between methylated and nonmethylated sequences. Msp I and Hpa II break the CCGG sequences. Nevertheless, Hpa II is ubable to break the DNA strand if the cytosine from the central nucleotide pair CG is methylated. In this work, Malpighian tubules from larvae from the last stage of M. quadrifasciata and M. rufiventris, subjected to the Feulgen reaction after by treatment with Msp I and Hpa II, were analysed in automatic scanning microspectrophotometry. Since a plasmal reaction was observed in this material, it was previously necessary the development of a methodological adjustement to make the Feulgen reaction specific to DNA. This was achieved by treatment of material with 5% sodium borohydrade followed by acetone-chloroform (1:1, v/v) before the Feulgen reaction. Also, although the definition of high and low heterochromatin content in Melipona after C-banding technique is not applicable to the chromatin of interphasic nuclei in Malpighian tubules of bees, it was demonstrated that DNA depurination in M. quadrifasciata was faster than that of M. rufiventris, thus confirming that this species has a higher condensed chromatin content. The Feulgen-DNA values for the heterochromatin of Melipona rufiventris, and for the heterochromatin besides some euchromatic domains of Melipona quadrifasciata, decreased after treatment with Msp I, remaining, however, unaltered after treatment with Hpa II. In conclusion, methylated CpG sequences may be part of different chromatin compartments, according to the considered species of the genus Melipona, and that their silencing effects may act by inducing the same cell physiology / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Many genetic processes require enzymes or enzyme complexes that interact simultaneously with distant sites along the genome. Such long-range DNA-enzyme interactions are important for example in gene regulation, DNA replication, repair and recombination. In addition many restriction enzymes depend on interactions between two recognition sites and form therefore a model system for studying long-range communications on DNA. Topic of the present work are Type III restriction enzymes. For these enzymes the communication mechanism between their distant target sites has not been resolved and conflicting models including 3D diffusion, 1D translocation and 1D diffusion have been proposed. Also the role of ATP hydrolysis by their superfamily 2 helicase domains which catalyse functions of many enzyme systems is still poorly understood. To cleave DNA, Type III restriction enzymes sense the relative orientation of their distant target sites and cleave DNA only if at least two of them are situated in an inverted repeat. This process strictly depends on ATP hydrolysis. The aim of this PhD thesis was to elucidate this long-range communication. For this a new single molecule assay was developed using a setup combining magnetic tweezers and objective-type total internal reflection fluorescence microscopy. In addition of being able to mechanically manipulate individual DNA molecules, this assay allows to directly visualize the binding and movement of fluorescently labelled enzymes along DNA. Applying this assay to quantum dot labelled Type III restriction enzymes, a 1D diffusion of the enzymes after binding at their target sites could be demonstrated. Furthermore, it was found that the diffusion depends on the nucleotide that is bound to the ATPase domains of these enzymes. This suggested that ATP hydrolysis acts as a switch to license diffusion from the target site which leads to cleavage. In addition to the direct visualization of the enzyme-DNA interaction, the cleavage site selection, the DNA end influence (open or blocked) and the DNA binding kinetics were measured in bulk solution assays (not part of this thesis). The experimental results were compared to Monte Carlo simulations of a diffusion-collision-model which is proposed as long-range communication in this thesis.

EcoP15I ist ein Vertreter der multifunktionalen, heterooligomeren Typ III Restriktionsendonukleasen. Typ III Restriktionsendonukleasen sind wegen der Lage ihres Spaltortes, ca. 25 bp vom Erkennungsort entfernt, von besonderem Interesse für Anwendungen in der Medizin und funktionellen Genomanalyse. EcoP15I erkennt die DNA-Sequenz 5''-CAGCAG und benötigt für eine effektive DNA-Spaltung zwei invers orientierte Erkennungsorte auf einem DNA-Molekül. Nach dem bisherigen DNA-Translokations-Modell bindet je ein EcoP15I-Protein an je einen Erkennungsort und startet dann durch ATP-Hydrolyse vermittelte DNA-Translokation. Die Kollision der beiden EcoP15I-DNA-Komplexe initiiert die DNA-Doppelstrang-Spaltung. Experimente zur Erkennungsort-Suche von EcoP15I zeigen, dass über längere Distanzen offenbar nicht das "Sliding", sondern ein dreidimensionaler Prozess die bevorzugte Bewegung von EcoP15I an der DNA ist. Eine erhöhte Anzahl von Wiederholungen von CAG-Trinukleotiden (CAG-Repeats) im Exon 1 des Gens für Chorea Huntington (Huntington Disease - HD) führt zur Manifestation dieser neurodegenerativen Erkrankung. Für die Diagnostik der Erkrankung ist die exakte Bestimmung der Anzahl der CAG-Repeats von Bedeutung. Diese Arbeit zeigt die Spaltung von HD Gen Exon 1 DNA durch EcoP15I. Die halbautomatische, hoch-sensitive Analyse dieses Spaltmusters ermöglicht die exakte Bestimmung der Anzahl der CAG-Repeats. Diese Arbeit liefert den ersten Nachweis für die DNA-Translokation durch eine Typ III-Restriktionsendonuklease. Die postulierten EcoP15I-DNA-Schlaufen wurden mit Hilfe der Rasterkraftmikroskopie (SFM) abgebildet. Dadurch wird das DNA-Translokations-Modell der DNA-Spaltung durch EcoP15I bestätigt. Es werden Gemeinsamkeiten und Unterschiede des gesamten DNA-Spaltvorganges der Typ III Restriktionsendonuklease EcoP15I in bezug auf andere Restriktionsendonukleasen diskutiert. / EcoP15I is a multifunctional, hetero-oligomeric Type III restriction enzyme. Type III restriction enzymes are of general interest in medicine and functional genome analysis because they cut DNA 25 bp downstream of their recognition site. EcoP15I recognises the DNA sequence 5`-CAGCAG and needs two inverse oriented recognition sites for effective DNA cleavage. According to the present translocation collision model DNA cleavage was proposed to result from ATP dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes. Experiments show that EcoP15 moves rather in a three-dimensional than in a "sliding" process in search for its recognition site. Huntington''s disease (HD) is a progressive neurodegenerative disorder with autosomal-dominant inheritance. The disease is caused by a CAG trinucleotide repeat expansion located in the first exon of the HD gene. To diagnose the illness the exact determination of the number of CAG repeats is necessary. This study shows that the number of CAG repeats in the HD gene can be determined by restriction of the DNA with the endonuclease EcoP15I and subsequent high-resolution analysis of the restriction fragment pattern using the ALFexpress DNA Analysis System. Here, for the first time DNA translocation by the Type III restriction enzyme EcoP15I is demonstrated. The postulated EcoP15-DNA loops are visualised using scanning force microscopy. This confirms the translocation-collision model for DNA cleavage by EcoP15. Similarities and differences between the DNA cleavage processes of the Type III restriction enzyme EcoP15I and other restriction enzymes are discussed.

EcoP15I gehört zu den heterooligomeren Typ III Restriktionsendonukleasen. Der multifunktionale Enzymkomplex ist aus zwei Modifikations- und zwei Restriktions-Untereinheiten aufgebaut und katalysiert sowohl die Spaltung als auch die Methylierung der DNA. Für die effektive Spaltung der doppelsträngigen DNA benötigt EcoP15I zwei invers orientierte Erkennungsorte mit der DNA-Sequenz 5’-CAGCAG. Die Spaltung erfolgt im oberen Strang 24 bis 26 Basen in 3’-Richtung nach dem Erkennungsort und im unteren Strang 26 bis 28 Basen in 5’-Richtung nach dem Erkennungsort. Aufgrund des bislang größten definierten Abstandes zwischen Erkennungs- und Spaltort ist EcoP15I ein wichtiges Werkzeug in der funktionellen Genomanalyse. Die Aufklärung der Domänenstruktur beider EcoP15I-Untereinheiten durch limitierte Proteolyse zeigte, dass die Restriktions-Untereinheit modular aufgebaut ist. Sie besteht aus zwei stabil gefalteten Domänen, der N-terminalen Translokase- und der C-terminalen Endo-Domäne. Beide Domänen sind durch einen flexiblen Linker verbunden. In der Modifikations-Untereinheit dagegen wurden keine Domänen identifiziert. Durch Insertion von Aminosäuren in und um den Linkerbereich konnten Enzymmutanten hergestellt werden, die bevorzugt die Positionen mit größten Abstand zum Erkennungsort spalteten. Wurden dagegen in dieser Region Aminosäuren deletiert, verloren die Enzymmutanten ihre DNA-Spaltaktivität. Die photochemische Vernetzung von EcoP15I mit spezifischer DNA ergab, dass EcoP15I drei Kontakte zum Phosphatrückgrat des ersten Adenins im Erkennungsort ausbildet. Ein Kontakt wird dabei über die Aminosäure S635 im C-terminalen Teil der Modifikations-Untereinheit hergestellt, zwei weitere entstehen durch die Aminosäuren Y248 und K421 der Restriktions-Untereinheit. Die transmissionselektronenmikroskopische Abbildung des negativ kontrastierten EcoP15I-Enzym zeigte einen symmetrischen Aufbau und stellt somit eine Grundlage für die Entwicklung eines dreidimensionalen Modells dar. / EcoP15I belongs to the hetero-oligemeric type III restriction endonucleases. The multifunctional enzyme complex consists of two modification and two restriction subunits and catalyses both the cleavage and methylation of the DNA. For effective cleavage of the double stranded DNA EcoP15I needs two inversely oriented recognition sites with the DNA sequence 5’-CAGCAG. The cleavage occurs 24 to 25 bases in 3’-direction from the recognition sequence in the top strand and 26 to 28 bases in 5’-direction from the recognition sequence in the bottom strand. Because of the largest known distance between recognition and cleavage site so far EcoP15I is an important tool in functional genomics. The elucidation of the domain structure of EcoP15I restriction as well as the modification subunit by limited proteolysis showed that the restriction subunit has a modular structure. It consists of two stable folded domains, the N-terminal translocase domain and the C-terminal endonuclease domain. Both domains are connected by a flexible linker. In contrast to the restriction subunit no domains could be detected in the modification subunit. Enzyme mutants that were constructed by insertion of amino acids in and around the linker region cleaved preferentially the position with the largest distance between recognition and cleavage site. The enzyme mutants lost their DNA cleavage activity when the amino acids in this region were deleted. The photochemical crosslinking of EcoP15I with specific DNA showed that EcoP15I forms three contacts to the phosphate backbone of the first adenine of the recognition site. One contact is made by amino acid S635 in the C-terminal part of the modification subunit. Two others are made by amino acids Y248 and K421 of the restriction subunit. The transmission electron microscope picture of the negatively stained EcoP15I enzyme showed a symmetric form and therefore it constitutes a basis for the development of a three dimensional model.

Die homodimere Typ IIE Restriktionsendonuklease EcoRII erfordert im Gegensatz zu den orthodoxen Typ II Restriktionsendonukleasen die simultane Wechselwirkung mit zwei Kopien ihrer DNA-Erkennungssequenz 5'CCWGG, um die spezifische endonukleolytisch Spaltung der DNA zu katalysieren. In der vorliegenden Arbeit wurde mittels Transmissionselektronenmikroskopie bewiesen, daß EcoRII die Bildung von DNA-Schlaufen an einem linearen DNA-Substrat mit zwei DNA-Erkennungsorten induziert - ähnlich wie andere DNA prozessierende Enzyme und Transkriptionsfaktoren. Kinetische Untersuchungen der DNA-Spaltreaktion von EcoRII mit superhelikaler Plasmid-DNA, die entweder einen oder zwei DNA-Erkennungsorte für EcoRII enthielt, zeigten, daß EcoRII pro Spaltereignis nur an einem der beiden involvierten doppelsträngigen DNA-Erkennungsorte spaltet. Die Studie, in der EcoRII photochemisch mit den Basen der DNA-Erkennungssequenz vernetzt wurde, ergab ein asymmetrisches Vernetzungsmuster, das durch die partielle Asymmetrie an der A/T-Position der ansonsten palindromischen Erkennungssequenz hervorgerufen wird. Wir konnten zeigen, daß die Aminosäure Tyr41 von EcoRII das 5'C des 5'CCAGG-Stranges der Erkennungssequenz kontaktiert. Durch Aufklärung der Domänenorganisation von EcoRII konnten wir das Modell der EcoRII-DNA-Interaktion verbessern. Wir zeigten, daß für die simultane Interaktion des Enzyms EcoRII mit zwei Kopien der Erkennungssequenz zwei verschiedene Domänen verantwortlich sind. Die C-terminale Domäne ist eine neue Restriktionsendonuklease, die effizienter als das vollständige EcoRII an einzelnen Erkennungsorten spaltet. Die N-terminale Domäne bindet spezifisch an die DNA und reduziert die Aktivität des vollständigen Enzyms, indem sie die Spaltung von einem zweiten Erkennungsort abhängig macht. Daher nehmen wir an, daß EcoRII in der Evolution in Form der N-terminalen Domäne eine zusätzliche DNA-Bindungsfunktion akquiriert hat, um eine neue Proteinfunktion zu entwickeln, die die Spaltung von DNA und die Interaktion mit zwei DNA-Erkennungsorten einschließt. Solche Interaktionen sind z.B. Voraussetzung für die DNA-Rekombination oder Transposition. Daher könnte die gegenwärtige EcoRII Restriktionsendonuklease eine evolutionärer Übergang von ortsspezifischen Endonukleasen zu einem neuen Protein sein, das spezifisch mit zwei DNA-Orten interagiert. / The homodimeric type IIE restriction endonuclease EcoRII requires the cooperative interaction with two copies of the recognition sequence 5'CCWGG for DNA cleavage. This is in contrast to the orthodox type II restriction endonucleases which interact with single recognition sequences. We have proven by transmission electron microscopy that EcoRII simultaneously binds two recognition sites on a linear DNA-substrate by looping out the intervening DNA. This DNA-loop formation is similar to that of other DNA processing enzymes and transcription factors. Kinetic investigations of the DNA cleavage of supercoiled DNA-plasmids containing either one or two recognition sites for EcoRII showed that EcoRII cleaves only at one of the two involved double-stranded DNA recognition sites. Photocross-linking of EcoRII to the bases of the recognition sequence revealed an asymmetric cross-linking pattern. This asymmetry is due to the partial asymmetry of the recognition sequence at the central A/T position. Furthermore, we found that amino acid Tyr41 of EcoRII specifically contacts the 5'C of the 5'CCAGG strand of the recognition sequence. We found by limited proteolysis that a two-domain structure enables EcoRII to interact cooperatively with two recognition sites. The C-terminal domain is a new restriction endonuclease that, in contrast to the complete EcoRII, specifically cleaves at single 5'CCWGG recognition sites. Moreover, this new restriction endonuclease cleaves much more efficiently than EcoRII. The N-terminal domain specifically binds the DNA-substrate and reduces the activity of EcoRII by making the enzyme dependent on a second recognition site. Therefore, we assume that a precursor EcoRII enzyme acquired another DNA binding domain to develop a new protein function that includes DNA cleavage and specific interaction with two DNA sites. The current EcoRII protein could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites such as DNA recombinases and transposases.

The EcoR124INT restriction-modification (R-M) system contains the genes HsdS3, HsdM and HsdR. S3 encodes the N-terminal domain of the wild-type S subunit and has been shown to dimerise in solution (Smith et al., 1998). Following purification of the subunits of the EcoR124INT R-M system, complexes of the methyltransferase S3/M and restriction endonuclease S3/M/R were formed and shown to have activity in vitro, methylating and hydrolysing a symmetrical DNA recognition sequence, respectively. The DNA mimic OCR (overcome classical restriction) protein inhibited the methyltransferase activity in vitro, with maximum inhibition at a 1: 2 molar ratio of (S3/M)2 to an ocr dimer. Dynamic light scattering (DLS), sedimentation equilibrium (SE) and sedimentation velocity (SV) experiments showed S3 to exist as a dimer and S11 (the central conserved domain of S) to exist as a tetramer in solution. M was found to be dimeric in solution, whilst the R protein was monomeric. A complex of S3/M was found to have a stoichiometry (S3/M)2 and a complex of S3/M/R had a stoichiometry of S3/M/R1, even when a 2: 1 molar ratio of R to S3/M, was added. Small angle neutron scattering (SANS) experiments provided values for the radius of gyration (Rg), which for S3 was comparable to that calculated for the recently published crystal structure of the S subunit from Methanococcus jannaschii (Kim et al., 2005). These experiments also showed a decrease in the Dmax in the presence of the 30 bp DNA recognition sequence from 200A to 140A, suggesting a similar conformational change in the positioning of the subunits as has been detected for the wild-type M. EcoR124I and a related type 1 1/2 system AhdI. This change following DNA binding was also observed by SV experiments. Furthermore ab initio modelling from the SANS data has provided a low-resolution structure for the EcoR124INT MTase and its complex with DNA.

Orientador: Clarice Weis Arns / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T02:18:52Z (GMT). No. of bitstreams: 1 Domingues_HelenaGallicchio_D.pdf: 1037559 bytes, checksum: 5f1aa70035e934b591bffbdd05c647b9 (MD5) Previous issue date: 2005 / Resumo: No presente estudo, técnicas para a detecção do vírus respiratório sincicial bovino, BRSV, visando ao diagnóstico e caracterização molecular deste patógeno, foram adaptadas e aplicadas em amostras coletadas de animais sem levar em consideração a presença de sinais e sintomas clínicos característicos de infecções causadas por esse agente. Foi coletado um total de 278 amostras de secreções nasais e fragmentos pulmonares de rebanhos bovinos provenientes dos estados de São Paulo e Rio Grande do Sul. Utilizando a técnica de RT-PCR detectou-se a presença de BRSV em sete amostras, duas de secreções nasais e cinco de fragmentos de pulmões. As amostras positivas foram submetidas ao isolamento viral e um novo isolado, denominado BRSV-108-BR, foi obtido após nove passagens em cultivos de células. Os fragmentos de 603 pb correspondentes ao segmento genômico da proteína G das amostras de BRSV em estudo, obtidos com a técnica de RT-PCR, foram submetidos à análise por enzimas de restrição-REA e ao seqüenciamento, visando sua caracterização molecular. Com a técnica de REA foram identificadas variações genéticas entre as amostras de BRSV detectadas, sugestivas de que duas amostras pertenciam ao subgrupo AB e cinco, ao subgrupo B de BRSV. Entretanto, a análise filogenética realizada pelo alinhamento das seqüências obtidas com seqüências disponíveis no GeneBank revelou que todas as amostras detectadas pertenciam ao subgrupo B. Com este estudo sugerimos que a técnica de REA possui utilidade limitada para classificação de BRSV em subgrupos, podendo ser utilizada como um instrumento prévio na caracterização das amostras, sendo, todavia, estritamente necessária uma análise baseada no seqüenciamento do gene da proteína G para caracterização de BRSV em subgrupos / Abstract: In this study techniques to detect the bovine respiratory syncytial virus (BRSV), aiming at the diagnosis and molecular characterization of this pathogen, where adapted and applied in samples collected from animals regardless of the presence of clinical signs and symptoms characteristic of infections caused by this agent. A total of 278 samples of nasal secretion and pulmonary fragments of bovine herds from the States of São Paulo and Rio Grande do Sul. By using the RT-PCR technique it was detected the presence of BRSV in seven samples, two of the nasal secretion samples, and five of the pulmonary fragments samples. The positive samples were submitted to viral isolation, and a new isolate named BRSV-108-BR was obtained after nine passages in cell cultivations. 603 pb fragments corresponding to the genomic segment of the G protein of the BRSV samples in study and obtained through the RT-PCR technique were submitted to restriction enzyme analysis (REA) and sequencing, aiming at their molecular characterization. With the REA technique, genetic variations were identified among the detected BRSV samples, suggesting that two samples belonged to the BSRV AB subgroup and five belonged to the BRSV B subgroup. However, phylogenetic analysis carried out by sequence alignment obtained with sequences available at the GeneBank revealed that all samples detected belonged to subgroup B. With this study we suggest that the REA technique to classify BRSV subgroups has a limited usefulness, serving only as a prior instrument to characterize the samples. Nevertheless, a subsequent analysis based on G protein sequencing is extremely necessary to characterize samples in one of the different BRSV existing subgroups / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular