Orientador: Nilson Ivo Tonin Zanchin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T17:37:36Z (GMT). No. of bitstreams: 1 Vaz_ThaisHaline_D.pdf: 13126521 bytes, checksum: 672f3c7b0345ee333ab24793e624068d (MD5) Previous issue date: 2008 / Resumo: A resposta individual das células está na base da resistência do organismo à infecção viral. O principal mecanismo de resistência envolve a participação de inúmeros genes da via de sinalização dos interferons. Vários estudos vêm sendo conduzidos em larga escala para identificar genes que respondem aos mais variados tratamentos, assim como clusters gênicos relacionados a determinadas enfermidades, como a leucemia. A função do produto de muitos destes genes ainda não foi caracterizada. Numa ampla revisão destes artigos identificamos a proteína KIAA0082/ISG95 respondendo a interferon, à infecção pelo vírus da hepatite C (HCV), ao tratamento celular com oligodeoxinucleotídeos CpG, fazendo parte de um cluster de genes relacionados à leucemia e sendo super-expressa em linfócitos T ativados. Embora não possua função conhecida, esta proteína apresenta quatro domínios que indicam uma possível atividade relacionada ao metabolismo de RNA. Neste trabalho demonstramos que o promotor do gene ISG95 responde à estimulação por interferon num sistema repórter em células Vero. As atividades bioquímicas de ISG95 foram determinadas usando a proteína recombinante expressa em células de inseto Sf9. ISG95 interage com RNA e com S-adenosilmetionina, possuindo também atividade de metiltransferase in vitro. Ensaios de localização sub-celular demonstraram sua distribuição nuclear. Além disso, através do método duplo-híbrido de levedura e de ensaio de co-imunoprecipitação, foi possível identificar sua interação com o domínio C-terminal (CTD) da RNA polimerase II, o que é consistente com sua localização nuclear e com a função predita para o domínio WW localizado na extremidade C-terminal de ISG95. Os resultados indicam que ISG95 é parte da via de resposta a interferon e tem função associada possivelmente a eventos de processamento de prémRNA mediados pelo domínio CTD da RNA polimerase II / Abstract: A major mechanism of cellular resistance to viral invasion involves genes from the interferon signaling pathway, called ISGs (interferon stimulated genes). Global transcriptional profiling studies have linked increased expression of ISG95 (KIAA0082) to response to interferon treatment and to viral infection, suggesting that it may be part of the cellular defense against viral replication. In this work, we shown that the ISG95 promoter can drive interferoninduced transcription of a reporter gene in Vero cell cultures. The biochemical functions of ISG95 were assessed using recombinant protein. ISG95 shows RNA- and S-adenosyl-methionine binding and protein methyltransferase activity in vitro. ISG95 interacts with the C-terminal domain of RNA polymerase II, which is consistent with its nuclear localization and with the predicted function of the WW domain found in the C-terminal region of ISG95. The results presented in this work indicate that ISG95 is part of the interferon response pathway and functions in the pre-mRNA processing events mediated by the C-terminal domain of the RNA polymerase II / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Septinas são proteínas ligantes a GTP encontradas desde fungos até metazoários. A primeira função identificada para septinas foi o seu papel central na organização e dinâmica do septo de divisão de leveduras. Uma das características marcantes é que septinas se organizam em heterofilamentos de 7 a 9 nm de espessura que foram purificados de diversos organismos tais como Saccharomyces cerevisiae, Drosophila e cérebro de camundongos. Hoje se sabe que septinas não estão envolvidas apenas nos processos de divisão celular, mas em uma variedade de processos como tráfico de vesículas, exocitose, interação com proteínas do citoesqueleto e com a membrana plasmática, o que resulta em alterações da morfologia celular. Neste trabalho foram desenvolvidos estudos da septina 4 humana (SEPT4) nos quais foi realizado a expressão e purificação da SEPT4 pelo uso do sistema de expressão heteróloga em E. coli e em células de insetos (Sf-9) via baculovírus. A tentativa de expressão usando o vetor pETTEV em E.coli não obteve sucesso, pois a proteína não foi expressa na forma solúvel. A construção do baculovírus recombinante AcSept4 e expressão da SEPT4 nas células de insetos foi realizada com êxito, mas o processo de purificação não foi satisfatório. Com o intuito de obter informações sobre possíveis proteínas que interagem com a SEPT4 e conseqüentemente sobre as funções desempenhadas por ela na célula, a SEPT4 foi utilizada como isca para ensaios de interação proteína-proteína pela técnica de duplo híbrido. Para isso, o gene da SEPT4 foi clonado fusionado ao domínio de ligação ao DNA Lex-A. A realização do ensaio de duplo híbrido com a proteína completa não foi possível, pois a mesma provocou a auto ativação do sistema, por isso uma nova construção foi realizada com a região GTPase e C-terminal SEPT4GC (124-478) como isca. Dentre as interações identificadas, foram encontradas apenas septinas do grupo II (SEPT6, SEPT8, SEPT10 e SEPT11) e quatro novas interações, que ainda precisam ser confirmadas. Por outro lado, uma interação já descrita na literatura envolve a proteína &#945-sinucleína, que é uma proteína abundantemente expressa no cérebro e associada à doença de Parkinson. O foco do estudo dessa interação foi realizar ensaios com os diferentes domínios da SEPT4 para comprovar uma interação direta e com isso tentar mapear o sítio de interação com a &#945-sinucleína. Os resultados obtidos pela ressonância plasmônica de superfície (SPR) indicam que o domínio C-terminal participa da interação com baixa afinidade (K,D=390 &#181M) e sugerem que o domínio GTPase também pode estar envolvido. Já os dados obtidos com os experimentos de RMN e anisotropia de fluorescência mostram indícios que a interação é dependente da conformação da &#945-sinucleína por que a interação aconteceria com maior afinidade quando a &#945-sinucleína está na presença de SDS. / Septins are a family of GTP binding proteins found in a great diversity of organisms. These proteins have been identified as having a central role in septum organization during yeast division. Septins are organized into heterofilaments which are 7 to 9 nm wide and these have been purified from yeast, Drosophila and mice brain. Septins are not only required for cell division, but seem to play a role also in vesicle trafficking and in the formation of diffusion barriers within cells, since they interact with cytoskeleton proteins and the plasma membrane causing changes in cell morphology. In the present work, the aim was investigate human Septin 4 (SEPT4), a septin highly expressed in the brain. One objective of this work was to find a suitable expression system and purification method for SEPT4. The protein was expressed in both E.coli and insect cells (Sf-9). Expression in E. coli with the vector pETTEV was unsuccessful because the protein was insoluble. Expression in insect cells using the recombinant baculovirus AcSept4, was obtained successfully, but the purification was difficult. Important information concerning SEPT4 function might be acquired, if interactions partners involved in cellular process were identified. With this goal in mind, a yeast two hybrid assays were performed. The sept4 gene was fused to the Lex-A DNA binding domain and used as bait in the yeast two hybrid essays. However, full length SEPT4 showed autonomous activation of reporter genes. A second construct was prepared including only GTPase domain and the carboxy terminus domain, (residues 124 to 478) and the screen of interactions were carried out only with SEPT4GC. All of the group II septins (SEPT6, SEPT8, SEPT10 and SEPT11) were identified together with four new interactions. The latter still need be confirmed. In addition, another interaction already described in the literature is between SEPT4 and &#945-synuclein, which is a protein highly expressed in brain and related to Parkinson\'s disease. Different spectroscopic methods and SPR were used to identify which domain of SEPT4 interacts directly with &#945-synuclein and in which region. The surface plasmon resonance (SPR) results indicate that the carboxy terminus participates in the interaction with low affinity (KD = 390 &#181M) and suggests that the GTPase domain may also be involved. The results obtained by fluorescence anisotropy and NMR studies provide evidence that the interaction is dependent on the &#945-synuclein conformation, because the affinity of SEPT4 and &#945-synuclein seemed to be higher in the presence of SDS.

Perceiving and responding to the environment is key to survival. Using the prokaryotic equivalent of a nervous system – the chemotaxis system – bacteria sense chemical stimuli and respond by adjusting their movement accordingly. In chemotactic bacteria, such as the well-studied E. coli, environmental nutrient sensing is achieved through a membrane embedded protein array that specifically clusters at the cell poles. Signalling to the motor is performed by activation of the CheA kinase, which phosphorylates CheY and CheB. CheY-P tunes the activity of the flagellar motor while CheB-P, together with CheR is involved in adaptation to the stimulus. In E. coli, a dedicated phosphatase terminates the signal. Most bacterial species however, have a much more complex chemotaxis network. Rhodobacter sphaeroides, a model organism for complex chemotaxis systems, has one membrane-embedded chemosensory array and one cytoplasmic chemosensory array, plus several homologs of the E. coli chemotaxis proteins. Signals from both arrays are integrated to control the rotation of a single start-stop flagellar motor. The phosphorelay network has been studied extensively through in vitro phosphotransfer while in vivo studies have established the components of each array and the requirements for formation. Mathematical modelling has also contributed towards inferring connectivities within the signalling network. Starting by constructing a two-hybrid-based interaction network focused on the components of the cytoplasmic chemosensory array, this thesis further addresses its associated adaptation network through a series of in vivo techniques. The swimming behaviour of series of deletion mutants involving the adaptation network of R. sphaeroides is characterised under steady state conditions as well as upon chemotactic stimulation. New connectivities within the R. sphaeroides chemotaxis network are inferred from analysing these data together with results from in vivo photoactivation localisation microscopy of CheB₂. The experimental results are used to propose a new model for chemotaxis in R. sphaeroides.

Le noyau est une innovation évolutive majeure caractéristique des organismes eucaryotes. Ces dernières années de nombreux travaux se sont intéressés à l’organisation de la chromatine dans l’espace nucléaire lors de l’interphase. Les protéines associées à la périphérie nucléaire ou ancrées dans la membrane nucléaire interne ont suscité un intérêt majeur due à leur contribution dans l’organisation spatiale de la chromatine. Chez les animaux, les lamines qui forment des filaments à la périphérie nucléaire et le complexe LINC, un complexe protéique reliant la membrane externe et interne du noyau sont connues pour interagir avec la chromatine, influencer l’organisation de cette dernière et moduler la régulation transcriptionnelle. Chez la plante modèle Arabidopsis thaliana utilisée dans ce travail, le complexe LINC est conservé, par contre les lamines ne le sont pas et seraient remplacées par d’autres acteurs spécifiques du règne végétal. Le travail détaillé dans ce manuscrit porte sur la mise en évidence d’un nouveau réseau d’interaction protéique localisé à la périphérie nucléaire et sur l’impact de ces protéines dans la morphologie du noyau et l’organisation de la chromatine. Mes travaux se sont concentrés sur les protéines à domaine SUN, l’une des composantes du complexe LINC et sur les protéines CRWN et KAKU4 présentes à la périphérie du noyau. Des cribles double hybride chez la levure m’ont permis d’identifier 24 partenaires protéiques potentiels dont plus d’un tiers sont des facteurs de transcription L’étude plus précise du facteur de transcription MaMYB pour lequel nous avons créé un allèle nul par la méthode CRISPR montre qu’il joue un rôle plus spécifique dans la formation des racines. L’étude de mutants combinatoires pour les gènes SUN, CRWN et KAKU4 montre des anomalies développementales notamment des tissus reproductifs. Enfin, une étude plus détaillée de la protéine KAKU4 suggère sa participation au maintien de la morphologie du noyau et au rapprochement de l’hétérochromatine vers la périphérie nucléaire. En résumé, mes travaux ont mis en évidence l’existence d’un réseau de facteurs de transcription recrutés à la périphérie nucléaire par les protéines SUN, CRWN et KAKU4. Ce réseau d’interaction protéine-protéine participerait à un mécanisme de séquestration de certains facteurs de transcription et/ou d'un rapprochement à la périphérie nucléaire de certains domaines de chromatine afin d’activer ou de réprimer leur transcription. / The nucleus is a major evolutionary innovation characteristic of eukaryotic organisms. In recent years, numerous studies have focused on the organization of chromatin in nuclear space during interphase. Proteins associated with the nuclear periphery or anchored in the inner nuclear membrane have been particularly studied for their contribution to the spatial organization of chromatin. In animals, the lamina that forms filaments at the nuclear periphery and the LINC complex, a protein complex linking the outer and inner membrane of the nucleus, are known to interact with chromatin, to influence its organization and to modulate transcriptional regulation. In the model plant Arabidopsis thaliana used in this work, the LINC complex is conserved, but not the lamina constituents, which are replaced by other specific actors of the plant kingdom. The work detailed in this manuscript identified a new protein interaction network located on the nuclear periphery and studied the impact of these proteins on nuclear morphology and chromatin organization. My work focused on SUN-domain proteins, one of the components of the LINC complex, and on the CRWN and KAKU4 proteins at the periphery of the nucleus. Double hybrid screens in yeast allowed me to identify 24 potential protein partners, more than a third of which are transcription factors. The more precise study of the transcription factor MaMYB for which we created a null allele using the CRISPR method, shows that it plays a more specific role in root formation. The study of mutant combinations for SUN, CRWN and KAKU4 genes reveals developmental abnormalities, particularly in reproductive tissue. Finally, a more detailed study of the role of the KAKU4 protein suggests that it contributes to the morphology of the nucleus in maintaining heterochromatin at the nuclear periphery. In summary, we propose the existence of a transcription factor network recruited to the nuclear periphery by SUN, CRWN and KAKU4 proteins. This protein-protein interaction network would participate in the sequestration of certain transcription factors and/or the localization of certain chromatin domains to the nuclear periphery in order to activate or suppress their transcription.

Exocyst is a binding protein complex, which is evolutionary conserved in yeast, animal and in plant cells. It has crucial role in regulation of cell morfogenesis and cell polarity. The function of the exocyst complex is binding of secretoric vesicle to the proper side on plasma membrane in penultimate step of exocytosis. This process is essecial for function and survival of cell. Another process crucial for the cell is autophagy. In plants autophagy plays important role in the responses to nutrient starvation, senescence, abiotic and biotic stress. RabG3b are small GTPases, which have positive role in autophagy. In this work I described the interaction between RabG3b and some of subunits of exocyst complex: Exo70B1, Exo70B2 and Exo84b. I also studied changes in morfogenesis of tonoplast by induction and inhibition of authophagy and induction of anthocyans synthesis in Arabidopsis thaliana.

Parkin is a cytosolic E3 ubiquitin ligase that is recruited to the mitochondria during cellular stress and has been suggested to be involved in a variety of biological processes such as mitophagy. The recruitment of Parkin (PARK2) to the mitochondria is dependent upon the kinase activity and the accumulation of PINK1 on damaged mitochondria. Mutations in either PINK1 or Parkin genes disrupt this protective pathway and lead to the accumulation of damaged mitochondria. From a clinical standpoint, mutations in the PARK2 gene have been associated with the progression and onset of autosomal recessive juvenile parkinsonism. Without the presence of a quality control system such as that of the PINK1/Parkin pathway, the accumulation of damaged mitochondria could lead to increased levels of oxidative stress, a decrease in ATP, and the progression towards cellular death. However, many of the details regarding the mechanism of Parkin-mediated ubiquitination and its involvement in mitophagy are not fully established. The intent of this thesis is to further explore the function of Parkin by utilizing the yeast-two hybrid system to identify novel Parkin interactors/substrates. A HeLa (cervical cell carcinoma) cDNA library was screened using Parkin124-465 as the "bait" protein. From this screening, six positive Parkin interactors were isolated and characterized. Using this approach it is possible to gain a better understanding of the function of Parkin in regulating cellular processes such as mitophagy.

Plants maintain organelle genomes that are descended from ancient microbes. Ages ago, these ancient microbes were engulfed by larger cells, beginning a process of co-evolution we now call the endo-symbiotic theory. Over time, DNA from the engulfed microbe was transferred to the genome of the larger engulfing cell, eventually losing the ability to be free-living, and establishing a permanent residency in the larger cell. Similarly, the larger cell came to rely so much on the microbe it had engulfed, that it too lost its ability to survive without it. Thus, mitochondria and plastids were born. Nearly all multicellular eukaryotes possess mitochondria; however, different evolutionary pressures have created drastically different genomes in plants versus animals. For one, animals have very compact, efficient mitochondrial genomes, with about 97% of the DNA coding for genes. These genomes are very consistent in size across different animal species. Plants, on the other hand, have mitochondrial genomes 10 to more than 100 times as large as animal mitochondrial genomes. Plants also use a variety of mechanisms to replicate and maintain their DNA. Central to these mechanisms are nuclear-encoded, organelle targeted replication proteins. To date, there are two DNA polymerases that have been identified in plant mitochondria and chloroplasts, Pol1A and Pol1B. There is also a DNA helicase-primase that localizes to mitochondria and chloroplasts called Twinkle, which has similarities to the gp4 protein from T7 phage. In this dissertation, we discuss the roles of the polymerases and the effects of mutating the Pol1A and Pol1B genes respectively. We show that organelle genome copy number decreases slightly and over time but with little effect on plant development. We also detail the interactions between Twinkle and Pol1A or Pol1B. Plants possess the same organellar proteins found in animal mitochondria, which are homologs to T7 phage DNA replication proteins. We show that similar to animals and some phage, plants utilize the same proteins in similar interactions to form the basis of a DNA replisome. However, we also show that plants mutated for Twinkle protein show no discernable growth defects, suggesting there are alternative replication mechanisms available to plant mitochondria that are not accessible in animals.

Thesis (MScMedSc (Biomedical Sciences. Medical Biochemistry))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: The genome of the pathogen Mycobacterium tuberculosis contains five copies of the ESAT-6 (ESX) gene cluster region, which encodes for a novel type VII secretion system. These gene cluster regions, which are directly involved in pathogenicity and phagosomal escape, contain genes encoding exported T-cell antigens ESAT-6 and CFP-10. The mechanism of action of the ESX secretion system however, remains largely unknown. This study focused on ESX gene cluster region 4 (ESX-4), which has been shown to be the most ancestral region and is also present in other species of Mycobacteria and even in other high G+C Gram-positive bacteria, such as Corynebacterium diptheriae and Streptomyces coelicolor. This project aimed to investigate the protein-protein interactions of ESX-4 of M. tuberculosis in the model organism Mycobacterium smegmatis by means of Mycobacterial Protein Fragment Complementation (M-PFC). M-PFC is a two-hybrid technique which employs two cloning vectors, pUAB300 (conferring resistance to hygromycin B) and pUAB400 (conferring resistance to kanamycin). Genes of interest are cloned into these vectors and co-transformed into the model organism M. smegmatis after which it is expressed as fusion proteins. Interaction of the proteins allows selective growth on a medium containing the antibiotic trimethoprim. Various interactions were identified throughout this region, including selfinteractions as well as the expected interaction between the ESAT-6 and CFP-10 protein family members esxT and esxU. Since this region is ancestral, ESX-4 provides the basic model of the mechanism of secretion of the type VII secretion system. Many similarities were apparent when the interactions identified for ESX-4 were compared to the interactions previously identified in ESX-3. Interactions identified by means of M-PFC provide a basis for the further study of the structure of this secretion system, and should be confirmed by means of other techniques, such as co-immunoprecipitation. Despite the ability of M-PFC to identify protein-protein interactions in a mycobacterial system, and thus overcoming some of the limitations of the classical yeast two-hybrid model, it must still be regarded as a fishing experiment for potential interactions. A further aim of the project was to construct a knock-out of ESX-4 in the model organism M. smegmatis, which contains three ESX regions, namely ESX-1, -3 and -4. Homologous recombination proved to be an effective technique for the construction of the knock-out, also indicating that ESX-4 is not essential for in vitro growth of M. smegmatis. The knock-out strain showed no morphological differences to the wild type strain of M. smegmatis. The knock-out strain will in future be compared to the wild type strain in various functional studies in order to determine the function of the ancestral ESX region. / AFRIKAANSE OPSOMMING: Die genoom van die patogeen Mycobacterium tuberculosis bavat vyf kopieë van die ESAT-6 geen groep gebiede wat kodeer vir ‘n unieke tipe VII sekresie sisteem. Die geen groep gebiede, wat direk betrokke is by patogenisiteit en fagosomale ontsnapping, bevat gene wat kodeer vir die gesekreteerde T-sel antigene ESAT-6 en CFP-10. Die meganisme van die ESX sekresie sisteem is egter steeds tot ‘n groot mate onbekend. Hierdie studie het gefokus op die ESX geen groep gebied 4 (ESX-4), wat voorheen bepaal is om die vroegste kopie van die gebied te wees en wat ook in ander species van Mikobakterieë en hoë G+C Gram-positiewe bakterieë, soos Corynebacterium diptheriae en Streptomyces coelicolor, voorkom. Hierdie projek was daarop gemik om die proteïen-proteïen interaksies van ESX-4 van M. tuberculosis in die model organisme Mycobacterium smegmatis te ondersoek deur middel van Mikobakteriële Proteïen Fragment Komplementasie (M-PFK). M-PFK is ‘n twee-hibried tegniek wat van twee kloningsvektore, naamlik pUAB300 (wat weerstand teen hygromycin B bied) en pUAB400 (wat weerstand teen kanamycin bied) gebruik maak. Gene van belang word in die vektore ingekloneer en in die model organisme, M. smegmatis geko-transformeer, waarna dit as fusieproteïene uitgedruk word. Indien ‘n interaksie tussen die proteïene plaasvind, sal selektiewe groei op ‘n medium wat die antibiotikum trimethoprim bevat, waargeneem word. Verskeie interaksies is in hierdie gebied geïdentifiseer, insluitende self-interaksies, sowel as die verwagte interaksie tussen die ESAT-6 en CFP-10 proteïen familielede esxT en esxU. Aangesien hierdie gebied die vroegste kopie is, bied ESX-4 die basiese model vir die meganisme van sekresie van die tipe VII sekresie sisteem. Wanneer interaksies wat vir ESX-4 geïdentifiseer is met die wat voorheen vir ESX-3 geïdentifiseer is vergelyk word is daar heelwat ooreenkomste. Interaksies wat deur middel van M-PFK geïdentifiseer is, verskaf ‘n basis vir die vêrdere studie van interaksies van hierdie gebied, en sal bevestig moet word deur gebruik te maak van aanvullende tegnieke, soos ko-immunopresipitasie. Ten spyte van die vermoë van M-PFK om proteïen-proteïen interaksies in ‘n mikobakteriële sisteem, wat dus sommige van die beperkings van die klassieke gis twee-hibriedmodel oorkom, te bestudeer, behoort dit steeds as ‘n voorlopige metode van identifikasie beskou te word. ‘n Vêrdere doel van die projek was om ‘n uitslaanmutant van ESX-4 in die model organisme M. smegmatis, wat drie van die ESX gebiede, naamlik ESX-1, -3 en -4 bevat, te skep. Homoloë rekombinasie is bewys om ‘n effektiewe tegniek te wees vir die skep van ‘n uitslaanmuntant en het daarop gedui dat ESX-4 nie essensieel is vir die in vitro groei van M. smegmatis nie. Die uitslaanstam het ook geen morfologiese verskille getoon teenoor die oorspronklike stam nie. Die uitslaanmutant sal in die toekoms gebruik word in ‘n verskeidenheid funksionele studies waar dit vergelyk sal word met die oorspronklike stam, ten einde die funksie van die vroegste ESX-gebied te bepaal. / Medical Research Council of South Africa / National Research Foundation of South Africa / Ernst and Ethel Eriksen Trust

No description available.

Chez les eucaryotes, la voie de protéolyse Ub/ protéasome 26S est responsable de la dégradation sélective de la plupart des protéines intracellulaires. Cette dégradation par le protéasome 26S est initiée par une polyubiquitination de la protéine réalisée grâce à l’action d’une cascade enzymatique impliquant 3 types d'enzymes nommées « ubiquitin-activating enzyme » (E1), « ubiquitin-conjugating enzyme » (E2) et « ubiquitin-protein ligase » (E3). Il existe différentes classes d’ubiquitines ligases (E3), parmi lesquelles la plus connue est le complexe SCF (Skp1-Cullin-F-box). La protéine SKP1 fixe à la fois la Culline et la F-box qui va reconnaitre spécifiquement la protéine cible. Contrairement aux protistes, les champignons et certains vertébrés qui possèdent un unique gène SKP1 fonctionnel, de nombreux animaux et espèces de plantes présentent plusieurs SKP1 homologues. Vingt et un et trente deux gènes SKP1 ont été décrits respectivement chez Arabidopsis thaliana et Oryza sativa. En dépit de l’importance du complexe SCF, chez le riz, peu de travaux décrivent les interactions entre les dizaines de protéines « SKP1-like » et les centaines de protéines F-box. Dans un premier temps, nous avons collecté et analysé les séquences de 288 gènes « SKP1-like » appartenant à 17 espèces, dont la mousse Physcomitrella patens, cinq monocotylédones et 11 eudicotylédones. Les analyses structurales et phylogénétiques de ces gènes indiquent qu’ils peuvent être divisés en différentes sous-familles. Nos analyses ont montré qu’OSK1 et OSK20 chez le riz constituent une classe de gènes SKP1 à intron unique conservé. Dans un deuxième temps, nous avons étudié le profil d’expression des gènes « SKP1-like » chez le riz. Notre investigation sur le nombre d’EST a montré que les gènes OSK1 et OSK20 sont les plus largement représentés dans les bases de données EST publiques. La méta-analyse de l’expression des gènes « SKP1-like » chez le riz, indique que les gènes OSK présentent des profils d'expression hétérogènes selon les tissus et les conditions physiologiques. Les résultats des intearctions protéine-protéine en double hybride ont révélé que les protéines OSK présentent différentes capacités d’interactions avec les protéines F-box. Cependant, OSK1 et OSK20 semblent interagir avec la plupart des protéines F-box testées. Les études de localisation subcellulaire ont indiqué que OSK1 et OSK20 sont des protéines nucléaires et cytosoliques. En se basant sur les divers résultats obtenus dans ce travail, nous pouvons suggérer que chez le riz, les gènes OSK1 et OSK20 sont fonctionnellement équivalents aux gènes ASK1 et ASK2 chez Arabidopsis thaliana. Nous pouvons également proposer les équivalents de ces gènes chez les autres espèces végétales dont le génome a été séquencé. / In eukaryotes, the ubiquitin Ub/26S proteasome pathway is responsible for the selective degradation of most intracellular proteins. This cellular process is initiated by protein polyubiquitination mediated by a three-step cascade involving: an ubiquitin-activating enzyme (E1), an ubiquitin-conjugating enzyme (E2) and an ubiquitin-protein ligase (E3). The E3 ubiquitin ligases contain several classes, among which the best-known are Skp1-Cullin-F-box (SCF) complexes. The SKP1 protein binds both Cullin and F-box which recognizes specifically the target proteins. Whereas protists, fungi and some vertebrates have a single functional SKP1 gene, many animal and plant species possess multiple SKP1 homologues. Twenty one and thirty-two SKP1-related genes have been described respectively in the Arabidopsis and Oryza sativa genome. Despite the importance of the SCF complex, there have been a few reports of systematic surveys of interactions between the dozens of SKP1-like proteins and the hundreds of F-box proteins in rice. In a first step, we retrieved and analyzed 288 SKP1-like genes belonging to 17 species including the moss Physcomitrella patens, five monocots and 11 eudicots. Structural and phylogenetic analysis of rice OSK genes and other plant SKP1-like genes have indicated that the different members of the plant SKP1 can be split into different subfamily. Our analyses indicated that OSK1 and OSK20 belong to a class of SKP1 genes that contain one intron at a conserved position. In a second step, we studied expression profiles of the rice Skp1-like genes. Our EST survey indicated that OSK1 and OSK20 are the most widely represented genes in public EST databases. Meta-analysis of the expression of rice SKP1-like genes indicated that OSK genes exhibit an expression profile that was heterogeneous in terms of tissues, conditions and overall intensity. Yeast two-hybrid results revealed that OSK proteins display a differing ability to interact with F-box proteins. However, OSK1 and OSK20 seemed to interact with most F-box proteins tested. Subcellular localization studies indicated that OSK1 and OSK20 are nuclear and cytosolic proteins. Based on the results obtained in this study, we can suggest that rice OSK1 and OSK20 are likely to have similar functions as do the Arabidopsis ASK1 and ASK2 genes. Similarly, we suggest a list of functional equivalent in the other sequenced plant genomes.