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Showing 31 to 40 of 40 (0.044 seconds)| 31 |
The regulation of chromosome segregation by Aurora kinase, protein phosphatase 1 and nucleolar protein UTp7Jwa, Miri 14 February 2012 (has links)
The Sli15-Ipl1-Bir1 chromosomal passenger complex is essential for proper kinetochore-microtubule attachment and spindle stability in the budding yeast Saccharomyces cerevisiae. Subcellular localization of this complex during anaphase is regulated by the Cdc14 protein phosphatase, which is kept inactive in the nucleolus until anaphase onset. I show here that the predominantly nucleolar ribosome biogenesis protein Utp7 is also present at kinetochores and is required for normal organization of kinetochore proteins and proper chromosome segregation. Utp7 associates with and regulates the localization of Sli15 and Cdc14. It prevents the abnormal localization of Sli15 on cytoplasmic microtubules, the premature concentration of Sli15 on the pre-anaphase spindle, and the premature nucleolar release of Cdc14 before anaphase onset. Utp7 regulates Sli15 localization not entirely through its effect on Cdc14. Furthermore, the mitotic exit block caused by Cdc14 inactivation is relieved partially by the simultaneous inactivation of Utp7. Thus, Utp7 is a multifunctional protein that plays essential roles in the vital cellular processes of ribosome biogenesis, chromosome segregation and cell cycle control. Protein phosphatase 1, Glc7 opposes in vivo functions of the Ipl1-Sli15-Bir1 kinase complex in budding yeast. I show here Scd5- a targeting subunit of Glc7 that regulates endocytosis/cortical actin organization and undergoes nuclear-cytoplasmic shuttling- is present at kinetochores. Ipl1 associates with both Glc7 and Scd5. The scd5-PP1[Delta]2 mutation, which disrupts the association between Glc7 and Scd5, also disrupts the association between Ipl1 and Scd5-Glc7 without affecting the kinetochore localization of these proteins. Genetic studies suggest that Scd5 may positively regulate both Glc7 phosphatase and the Ipl1 kinase complex. In accordance, Scd5 stimulates in vitro kinase activity of Ipl1. scd5-PP1[Delta]2 cells missegregate chromosomes severely due to several defects: i) at least one of sister kinetochores appears not attached to microtubule. ii) sister chromatids are persistently cohesed through anaphase. iii) Sli15 is hyperphosphorylated and less abundant on the anaphase spindle resulting in unstable mitotic spindle. These results together suggest that Scd5 functions in diverse processes that are essential for faithful chromosome segregation. How Scd5 coordinately regulates two apparently antagonistic enzymatic activities of Ipl1 and Glc7 remains to be determined. / text
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Adding gears to the RNA machine: discovery and characterisation of new classes of small RNAs in eukaryotesRyan Taft Unknown Date (has links)
Genome sequencing has yielded unparalleled insights into fundamental biological processes and the genetics that guide them. In contrast to expectations that protein-coding genes would be the primary output of eukaryotic genomes, however, it is now clear that the vast majority of transcription is devoted to noncoding RNAs (ncRNAs). Although originally regarded as 'transcriptional noise', it is now clear that these transcripts are essential regulators of genetic activity. In this thesis I build upon the hypothesis that the genomes of eukaryotes encode a regulatory 'RNA machine' dominated by ncRNAs. In the Introduction (Chapter 1) I discuss how prior gene models may have inadvertently prevented a full understanding of ncRNAs, review the transcriptional landscape of eukaryotes, and examine the biogenesis and function of small regulatory RNAs. In support of a role for ncRNAs in complex metazoa, Chapter 2 presents data showing a positive correlation between the proportion of non-protein-coding DNA and biological complexity, suggesting that the evolutionary trajectory of intricate developmental phenotypes may have been facilitated by ncRNAs. In the following chapters two more 'gears' are added to the RNA machine. Chapter 3 details the discovery of snoRNA-derived RNAs - an evolutionarily ancient class of Argonaute-assocaited RNA whose biogenesis overlaps with microRNAs (miRNAs) and silencing RNAs (siRNAs). Likewise, Chapter 4 reports a new class of ~18 nt transcription initiation RNAs (tiRNAs) derived from regions proximal to transcription start sites. tiRNAs are enriched at GC-rich promoters and regions of active transcription, implicating them in transcriptional regulation. Chapter 5 presents evidence that tiRNAs are restricted to metazoa, and describes a model of RNA Polymerase II dependent tiRNA biogenesis. This thesis concludes with a general discussion of the implications of these findings, and the potential development of RNA therapeutics. Gathering evidence suggests that eukaryotic genomes are driven by a complex and interwoven network of RNA regulatory feedback loops. This thesis takes a small step towards developing a complete picture of this system.
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Adding gears to the RNA machine: discovery and characterisation of new classes of small RNAs in eukaryotesRyan Taft Unknown Date (has links)
Genome sequencing has yielded unparalleled insights into fundamental biological processes and the genetics that guide them. In contrast to expectations that protein-coding genes would be the primary output of eukaryotic genomes, however, it is now clear that the vast majority of transcription is devoted to noncoding RNAs (ncRNAs). Although originally regarded as 'transcriptional noise', it is now clear that these transcripts are essential regulators of genetic activity. In this thesis I build upon the hypothesis that the genomes of eukaryotes encode a regulatory 'RNA machine' dominated by ncRNAs. In the Introduction (Chapter 1) I discuss how prior gene models may have inadvertently prevented a full understanding of ncRNAs, review the transcriptional landscape of eukaryotes, and examine the biogenesis and function of small regulatory RNAs. In support of a role for ncRNAs in complex metazoa, Chapter 2 presents data showing a positive correlation between the proportion of non-protein-coding DNA and biological complexity, suggesting that the evolutionary trajectory of intricate developmental phenotypes may have been facilitated by ncRNAs. In the following chapters two more 'gears' are added to the RNA machine. Chapter 3 details the discovery of snoRNA-derived RNAs - an evolutionarily ancient class of Argonaute-assocaited RNA whose biogenesis overlaps with microRNAs (miRNAs) and silencing RNAs (siRNAs). Likewise, Chapter 4 reports a new class of ~18 nt transcription initiation RNAs (tiRNAs) derived from regions proximal to transcription start sites. tiRNAs are enriched at GC-rich promoters and regions of active transcription, implicating them in transcriptional regulation. Chapter 5 presents evidence that tiRNAs are restricted to metazoa, and describes a model of RNA Polymerase II dependent tiRNA biogenesis. This thesis concludes with a general discussion of the implications of these findings, and the potential development of RNA therapeutics. Gathering evidence suggests that eukaryotic genomes are driven by a complex and interwoven network of RNA regulatory feedback loops. This thesis takes a small step towards developing a complete picture of this system.
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Estudo comparativo das características citogenéticas e moleculares de Triatoma maculata e Triatoma pseudomaculata (Triatominae, Heteroptera)Mendonça, Priscila Pasquetto [UNESP] 19 February 2010 (has links) (PDF)
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mendonca_pp_me_sjrp.pdf: 681812 bytes, checksum: d7353da4e0742a18d8d505b0587fda7b (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Os triatomíneos são insetos hematófagos de grande importância para a parasitologia humana, pois são transmissores do Trypanosoma cruzi, protozoário causador da doença de Chagas. Além de sua importância médico-sanitária, os triatomíneos destacam-se pela sua citogenética, pois possuem cromossomos holocêntricos e um modelo de meiose incomum, com meiose invertida para os cromossomos sexuais. Recentes pesquisas com marcadores moleculares em triatomíneos tentam compreender a ancestralidade do grupo. Uma das formas de se compreender a evolução entre espécies é a partir da análise de seqüências do DNA ribossômico (DNAr). Por pertencer a famílias multigênicas, cópias individuais do DNAr não acumulam mutações independentemente, resultando em pequena variação intra-específica e relevante diferenciação interespecífica. No presente trabalho foi realizado um estudo comparativo entre as espécies Triatoma maculata e Triatoma pseudomaculata, com base no uso das técnicas citogenéticas convencionais de orceína lacto-acética, impregnação por íons prata, bandamento-C, reação de Feulgen; da técnica de citogenética molecular de bandamento- C CMA/DAPI; e também por meio da análise da região ITS-1 do DNAr, com base no sequenciamento, com o objetivo de avaliar o grau de homologia entre as espécies estudadas. Os cariogramas das duas espécies indicaram dez pares de autossomos (um deles de tamanho maior) e um par de cromossomos sexuais (2n = 22). No ciclo meiótico foi possível observar a fragmentação da região nucleolar no final do estágio difuso. Corpúsculos nucleolares foram observados em alguns dos núcleos em metáfases meióticas de T. pseudomaculata, evidenciando a persistência nucleolar. A técnica de bandamento-C revelou que o cromossomo Y é heterocromático em ambas as espécies. O sequencimento da região ITS-1, indicou que as espécies apresenta... / The triatomines are hematophagous insects of great concern in public health because they are vectors of Trypanosoma cruzi, a protozoan that causes Chagas disease. Triatomines are also of great genetic interest, because that they present holocentric chromosomes and an unusual form of meiosis with post-reductional segregation of sex chromosomes. Recent studies based on molecular markers try to understand the evolutionary history of triatomines. To understand the evolution of a given species, ribosomal DNA (rDNA) analyses are frequently used, which can help to infer evolutionary relationships among species. Individual copies of rDNA do not accumulate mutations independently because they belong to multigene families, resulting in slight intraspecific and important interspecific variation. In this study, a comparative analysis was performed between the species Triatoma maculata and Triatoma pseudomaculata, based on the cytogenetic techniques of lacto-acetic orcein, silver ion impregnation, Cbanding, Feulgen reaction; and CMA/DAPI C-banding. We also compared the species by sequencing the ITS-1 rDNA internal transcribed region in order to evaluate the degree of homology among the studied species. The cariograms of the two species revealed ten autosomes and one pair of sexual chromosomes (2n= 22). In the meiotic cycle, nucleolar fragmentation during the final stages of meiotic prophase I was found. Nucleolar corpuscles were found in some meiotic metaphases of T. pseudomaculata, which is evidence of nucleolar persistence. The C-banding technique revealed that the Y chromosome is heterochromatic in both species. The ITS-1 rDNA sequences showed that the species presented a discharge proximity to each other, and had a high degree of homology (98.5%). The knowledge obtained in this study contributes to the understanding of the interrelation and distribution of those species, and offers... (Complete abstract click electronic access below)
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Vitelogeneze karyofylidních tasemnic. / Vitellogenesis in caryophyllidean cestodes.DROBNÍKOVÁ, Petra January 2010 (has links)
Vitellogenesis in two caryophyllidean cestodes Caryophyllaeus laticeps and Khawia sinensis, parasitizing cyprinid fishes, were examined using light(LM)and transmission electron microscopy(TEM)and cytochemical staining for glycogen. Mature vitelline folicles consist of vitelline cells at various stages of development and an interstitial tissue. Maturing and mature vitellocytes contain vitelline material in the form of single small shell globules, which fuse and give rise to the large shell globule clusters.Glycogen was present in the cytoplasm and in the nucleus of the mature vitellocytes. Small lipid droplets were found in the cytoplasm of C. laticeps. "Lamellar granules" were observed in the cytoplasm of the mature vitellocytes in K. sinensis.
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Caracterização do ciclo nucleolar e da formação do corpo cromatóide na espermatogênese de alguns vertebrados /Peruquetti, Rita Luiza. January 2009 (has links)
Orientador: Maria Tercília Vilela de Azeredo Oliveira / Banca: Maria Luiza Silveira Mello / Banca: Reinaldo Azoubel / Banca: Carlos Alberto Vicentini / Banca: Eliana Morielle Versute / Resumo: O corpo cromatóide (CB) é uma organela citoplasmática que, aparentemente, possui um papel no estoque de RNA e proteínas para a diferenciação final dos espermatozóides. Existem algumas teorias que tentam explicar a origem do material que compõe essa organela. Uma dessas teorias, proposta por alguns autores, sugere que o CB se origine a partir de material nucleolar, que se fragmenta nas etapas iniciais da espermatogênese e, em seguida, migra para o citoplasma. O objetivo do presente estudo foi acompanhar o ciclo nucleolar por meio de análises citoquímicas - hematoxilina-eosina (HE); azul de toluidina (AT); variante da concentração crítica de eletrólitos (CEC); reação de Feulgen; impregnação por íons prata (AgNOR); citogenéticas - impregnação por íons prata (AgNOR), e análises ultra-estruturais - microscopia eletrônica de transmissão (MET), para verificar a relação da fragmentação do material nucleolar com a formação do corpo cromatóide (CB), em algumas espécies de vertebrados: Tilapia rendalli (Teleostei, Cichlidae); Dendropsophus minutus (Amphibia, Anura); Phrynops geoffroanus (Reptilia, Testudines) e coelho albino da raça Nova Zelândia - Oryctolagus cuniculus (Mammalia, Lagomorpha). Por meio das análises citoquímicas foi possível observar que ocorre uma fragmentação do material nucleolar no início da prófase I, em todas as espécies analisadas, e uma posterior reorganização do nucléolo no núcleo de espermátides iniciais, com uma área significantemente menor do que a área do nucléolo das espermatogônias. Três fenômenos podem contribuir para essa diferença significante entre as áreas nucleolar de espermatogônias e espermátides: a) Modificação no estado funcional da célula; b) Diminuição no número de RONs nas espermátides; c) Migração de material nucleolar fragmentado ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The chromatoid body (CB) is a cytoplasmic organelle that has a function related to RNA and protein accumulation and ⁄ or storage for later germ-cell differentiation. Many theories have been postulated in order to explain the origins of the CB material. One of the most accepted theory describes that it originates from a nucleolar material, where it was fragmented in the early spermatogenesis, and finally, this fragmented nucleolar material migrates to cytoplasm. The aims of the present study were: 1) monitoring the nucleolar material distribution by means of cytochemical techniques (hematoxylin-eosin (HE), toluidine blue (TB), modified Critical Electrolyte Concentration for detecting RNA (CEC), silver-ion impregnation (AgNOR) and Feulgen reaction), and by ultrastructural analysis (Transmission Electron Microscopy - TEM); and 2) comparing the nucleolar material distribution with the formation of CB in some vertebrate species: Tilapia rendalli (Teleostei, Cichlidae); Dendropsophus minutus (Amphibia, Anura); Phrynops geoffroanus (Reptilia, Testudines); and Oryctolagus cuniculus (Mammalia, Lagomorpha). For all analyzed species, the cytochemical techniques showed that the nucleolar fragmentation occurred during the beginning of prophase I, and the nucleolus reorganization occurred in the early spermatids nucleus. Statistical tests evidenced that area of the early spermatids nucleolus were smaller than the spermatogonia nucleolus area. Three phenomena can contribute for the statistical difference between the spermatogonia nucleolar area and the early spermatids nucleolar area: a) Modification of cell activity; b) Decrease of the number of NORs in the spermatids; c) Migration of the fragmented nucleolar material from the nucleus to the cytoplasm. This nucleolar material will participate in the CB formation process. The ultrastructural analysis showed an ...(Complete abstract click electronic access below) / Doutor
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Characterization of the MDM2 binding regions of ribosomal protein L5Plummer, Kevin D. 20 July 2010 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / The MDM2-p53 feedback loop is a well-characterized pathway. p53 is a
transcription factor and regulates the transcriptional expression of genes that
encode proteins responsible for cellular senescence, cell cycle arrest,
apoptosis, and DNA repair. Various cellular stresses can result in p53
activation, including hypoxia, DNA damage by agents such as UV or IR,
oncogenic signaling, nucleotide depletion and nucleolar stress from
perturbation of ribosomal biogenesis. Under normal conditions, MDM2’s role in
the pathway is to inhibit p53 function by directly binding to this protein and
facilitating its ubiquitylation and 26S proteasome-mediated degradation.
Under stressful cellular conditions, certain proteins interact with and rescue
MDM2’s inhibition of p53. For example, upon exposure to small amounts of
Actinomycin D, rRNA transcript synthesis is stalled resulting in the release of
various ribosomal proteins including RPL5, RPL11 and RPL23; each of which
has been shown to bind MDM2 within its central acidic domain and inhibit its
ability to destabilize p53. Although the RPL5 binding region of MDM2 have
been mapped in prior investigations, the MDM2-binding region(s) of RPL5
have yet to be characterized.
By employing RPL5 deletion mutagenesis and in vitro GST-fusion
protein-protein association assays with purified proteins, this dissertation
attempts to elucidate those regions of RPL5 that may interact with MDM2.
Normalizing RPL5-WT to 1.00, our study reveals that the basic N and C-terminals
of RPL5 appear to bind with MDM2 while RPL5’s central region
displays negligible binding to the central acidic domain of MDM2. Also, the
possible meanings of these RPL5 MDM2 binding domains are discussed along
with their utilization in potential future applications.
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Role PML v ribosomálním stresu / Role of PML in ribosomal stressKremserová, Petra January 2019 (has links)
PML is involved in many cellular processes. It organizes nuclear structures PML nuclear bodies (PML NBs) and it associates with nucleolus in response to ribosomal stress to form PML nucleolar associations (PNAs). The function of PNAs is unclear. To elucidate this question, one can attempt to identify proteins interacting with PML at nucleolus. The common method is co- immunoprecipitation, however, this approach cannot be used for PML due to its low solubility. To defeat this, an alternative way of proximity-dependent biotin labelling could be used. The goal of this work was to explore a suitability of biotin labelling for identification of PML nucleolar partners. For this purpose I prepared constructs of wild type or mutated PML with GFP and biotin ligase for transient and stable expression and analysed their propensity to form PML NBs and doxorubicin-induced PNAs, and biotinylate their vicinity. In transient expression, both fusion proteins formed PML NBs and only wild type but not mutated PML IV formed PNAs after doxorubicin treatment with preserved biotinylation capability. In stable expression of fusion proteins in cells with PML knockout the number and composition of PML NBs was aberrant and no PNAs were observed. However, this system was utilized for optimization of solubilisation of biotinylated...
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Analýza karyotypu u vybraných bičovců řádů Amblypygi a Uropygi / Karyotype analysis of selected representatives of two pedipalpid orders, Amblypygi and UropygiSember, Alexandr January 2010 (has links)
Karyotype analysis of selected species from arachnid orders Amblypygi and Uropygi Whip spiders (Amblypygi) and whip scorpions (Uropygi) represent relict arachnid orders which has been found already at Upper Carboniferous strata. Although cytogenetic data from amblypygids and uropygids might be important to reconstruct karyotype evolution of arachnids, cytogenetics of these orders is almost unknown. Presented study is aimed in analysis of karyotype and meiosis in 16 species of Amblypygi and 4 species of Uropygi. Both groups are characterized by considerable range of diploid chromosome numbers (2n = 24 - 86 in Amblypygi and 36 - 66 in Uropygi). Analysed species does not exhibit morfologically differentiated sex chromosomes. Differentiation of sex chromosomes on molecular level was revealed in amblypygid Paraphrynus mexicanus by comparative genome hybridization. Obtained data indicate XY/XX sex chromosome system in this species. Comparison of karyotype data indicates reduction of chromosome numbers during evolution of both orders. In Amblypygi, this reduction was accompanied by increase of number of biarmed chromosomes. This trend is not apparent in Uropygi. Karyotypes of most analysed amblypygids and uropygids are also characterized by low amount of heterochromatin. Most studied species exhibit two pairs...
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SCF cdc4 regulates msn2 and msn4 dependent gene expression to counteract hog1 induced lethalityVendrell Arasa, Alexandre 16 January 2009 (has links)
L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.
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