L'environnement des organismes vivants est par définition fluctuant, toutes variations aléatoires du milieu de vie constituent un stress pour les cellules. Au fil de l'évolution, une forte pression de sélection a façonné le fonctionnement cellulaire jusqu'aux réponses complexes élaborées par les organismes vivants. Mes travaux s'inscrivent autour des mécanismes moléculaires de la réponse au stress et plus particulièrement les stress génotoxiques. La première partie de l'étude décrit finement la réplication de l'ADN en condition de stress réplicatif. Ainsi, nous avons montré que les pools de dNTPs sont limitants pour la progression des fourches de réplication en phase S normale et en stress, et que leurs niveaux conditionnent le programme temporel de réplication. De plus, nous avons mis en évidence un mécanisme d'adaptation au stress réplicatif et aux dommages constitutifs dans des mutants caractérisés par de l'instabilité génétique (CIN) via l'activation du checkpoint de dommage conduisant à l'expansion des pools de dNTPs. Pour finir, nous montrons que l'augmentation des niveaux de dNTPs facilite la réplication en présence de lésions de l'ADN, d'une manière indépendante des ADN polymérases translésionnelles. Le second projet apporte de nouveaux éléments sur le rôle de Crt10 in vivo, préalablement identifié comme un régulateur transcriptionnel des gènes de la Ribonucléotide Réductase (RNR). Nos données indiquent que les mutants crt10Δ ont des niveaux de dNTP similaires à ceux des cellules sauvages, et que cette mutation a un très faible impact sur l'expression des gènes RNR, malgré un phénotype de vitesse de progression des fourches accrue. Nous montrons que le mutant crt10Δ est caractérisé par un défaut d'entrée en phase S et d'initiation des origines de réplication. L'origine de ce défaut pourrait résider dans les fonctions de Crt10 impliquant la régulation de la biosynthèse des ribosomes au sein du complexe Rtt101-Mms1. Le troisième projet identifie MRX (Mre11-Rad50-Xrs2) comme un acteur de la voie de terminaison des ARN non codants. MRX s'associe à des loci recrutant également le complexe de terminaison Nrd1-Nab3-Sen1 à l'échelle du génome entier. L'inactivation de RAD50 se traduit par une perte d'efficacité de terminaison et l'accumulation de transcrits bicistroniques, ainsi qu'une dérégulation du niveau d'ARNs non codants instables (CUT) et de leurs gènes associés. Tout comme Sen1, MRX pourrait intervenir dans la résolution des collisions entre les machineries de transcription et de réplication. / A fluctuating environment is a powerful mean of selection for living organisms, which evolved complex signaling networks to integrate these variations and direct swift and efficient cellular responses. The aim of my work is the identification and characterization of molecular mechanisms involved in the tolerance of replicative stress and DNA damage. First, we show that changes in dNTP pools affect several aspects of replication dynamics in budding yeast. dNTP levels are limiting for normal S-phase progression and determine the temporal program of replication during a replicative stress. Interestingly, we also observed that chromosomal instability (CIN) mutants display expanded dNTP pools due to the constitutive activation of the DNA damage checkpoint. Since increased dNTP levels promote forks progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replicative stress by upregulating dNTP pools. Secondly, we bring new lights on the role of Crt10 in vivo, which has been initially identified as a negative regulator of Ribonucleotide Reductase (RNR) genes expression. Deletion of CRT10 neither leads to expanded dNTP pools, nor to a massive deregulation of RNR genes, although crt10Δ cells exhibit faster fork progression. The crt10Δ mutant accumulates at the G1/S transition and exhibits a strong defect of origin firing that could account for its replication phenotype. Moreover, we observed a global decrease in ribosome biogenesis in crt10Δ. The physical interaction of Crt10 with several members of the ribosome biogenesis pathway and its role in the Rtt101-Mms1 complex suggest that Crt10 may regulate ribosome levels in vivo. At last, we identified MRX (Mre11-Rad50-Xrs2) as a bona fide member of the transcription termination of non-coding RNA (ncRNA). ChIP-seq reveals that MRX localized at the same loci than the Nrd1-Nab3-Sen1 complex in vegetative growth. rad50Δ cells exhibit transcriptional read-through and upregulation of unstable cryptic transcripts (CUTs) leading to a misregulation of their associated gene. Finally, MRX seems to be involved in the resolution of branched structures emanating from collision between transcription and replication machineries, as it is the case for Sen1.

Les cassures double brin de l’ADN (CDB) sont des lésions qui peuvent conduire à des réarrangements génétiques. Deux voies sont impliquées dans la réparation de ces dommages: la recombinaison homologue (HR) et la ligature d’extrémités nonhomologues (NHEJ).Au laboratoire un substrat intrachromosomique permettant de mesurer l’efficacité et la fidélité du NHEJ à été mis en place (Guirouilh-Barbat 2004). Cette approche a permis de démontrer l’existence d’une voie alternative à KU qui utilise des microhomologies présentes de part et d’autre de la cassure - le NHEJ alternatif (Guirouilh-Barbat 2004, Guirouilh-Barbat et Rass 2007). Les travaux de ma thèse consistent à caractériser les principaux acteurs de cette voie. En absence de KU, cette voie alternative du NHEJ, s'initierait tout d’abord parla résection d'extrémités d’ADN non protégées. Nous avons montré que l’activité nucléasique de MRE11 est nécessaire à ce mécanisme. La surexpression de MRE11 conduit à une stimulation du NHEJ, contrairement à l’extinction de la protéine par siRNA, résultant en une baisse de son efficacité de deux fois. Nos résultats montrent également que les protéines RAD50 et CtIP agissent dans la même voie que MRE11. De plus, dans les cellules déficientes pour XRCC4, la MIRIN – un inhibiteur du complexe MRN - conduit à une chute de l'efficacité de la réparation, démontrant le rôle de MRE11 dans la voie alternative du NHEJ. Nous avons aussi montré que MRE11 peut agir de manière dépendante et indépendante de la kinase ATM (Rass et Grabarz, Nat Struct Mol Biol 2009). L'initiation de la résection de la cassure doit être ensuite poursuivie par une dégradation plus importante de l'ADN qui est assuré par les protéines Exo1 et Sgs1/Dna2 chez la levure. Chez les mammifères, des études in vitro suggèrent un modèle similaire à deux étapes. Nous avons choisi de nous intéresser au rôle de la protéine BLM, qui est l’un des homologues humains de la RecQ hélicase Sgs1, dans la résection. Nos expériences montrent que l’absence de BLM diminue l’efficacité du NHEJ. De plus, l’extinction de BLM conduit à une augmentation d’évènements infidèles lors de la réparation par NHEJ et l’apparition d’évènements de résection de grande taille (>200nt). Ceci suggère que BLM protège contre de longues résections lors de la mise en place du NHEJ alternatif. De manière cohérente, BLM est impliquée dans la protection contre la résection dépendante de CtIP lors des étapes précoces de la recombinaison homologue. En conclusion, nos résultats montrent un rôle prédominant de BLM dans la protection contre un excès de résection médiée par CtIP. BLM interagit avec 53BP1 aux sites de dommages de manière dépendante d’ATM afin de réguler le processus de résection, en contrecarrant l’action de BRCA1. Ceci souligne à nouveau le rôle essentiel de BLM dans la protection contre la résection et la favorisation de la conversion génique sans crossing-over, ce qui est primordial pour le maintien de la stabilité du génome. / DNA double strand breaks (DSBs) are highly cytotoxic lesions, which can lead to genetic rearrangements. Two pathways are responsible for repairing these lesions : homologous recombination (HR) and non homologous end joining (NHEJ). In our laboratory, an intrachromosomal substrate has been established in order to measure the efficiency and the fidelity of NHEJ in living cells (Guirouilh-Barbat 2004). This approach led us to identify a KU-independent alternative pathway, which uses microhomologies in the proximity of the junction to accomplish repair – the alternative NHEJ (Guirouilh-Barbat 2004, Guirouilh-Barbat et Rass 2007). The goal of my thesis consisted in identifying and characterising major actors of this pathway. In the absence of KU, alternative NHEJ would be initiated by ssDNA resection of damaged ends. We showed that the nuclease activity of MRE11 is necessary for this mechanism. MRE11 overexpression leads to a two fold stimulation of NHEJ efficiency, while the extinction of MRE11 by siRNA results in a two fold decrease. Our results demonstrate that the proteins RAD50 and CtIP act in the same pathway as MRE11. Moreover, in cells deficient for XRCC4, MIRIN – an inhibitor of the MRN complex – leads to a decrease in repair efficiency, implicating MRE11 in alternative NHEJ. We also showed that MRE11 can act in an ATM-dependent and independent manner (Rass et Grabarz Nat Struct Mol Biol 2009). The initiation of break resection needs to be pursued by a more extensive degradation of DNA, which is accomplished in yeast by the proteins Exo1 and Sgs1/Dna2. In human cells, in vitro studies have recently proposed a similar model of a two-step break resection. We chose to elucidate the role of one of the human homologs of Sgs1 – the RecQ helicase BLM – in the resection process. Our experiments show, that he absence of BLM decreases the efficiency of end joining by NHEJ, accompanied by an increase in error-prone events, especially long-range deletions (>200nt). This suggests that BLM protects against extensive resection during alternative NHEJ. Furthermore, BLM is implicated in the protection against CtIP-dependent resection at the initiation of HR. In conclusion, our results show a major role of BLM in protecting against an excess of resection, mediated by the MRN cofactor – CtIP. BLM interacts with 53BP1 at sites of damage, in an ATM-dependent manner, in order to regulate the resection process and counteract BRCA1 activity. This underlines the novel role of BLM in the protection against resection and favouring gene conversion events without crossing-over, which is substantial for maintaining genomic integrity.

Over the course of an organism's life, its genome is exposed to endogenous and exogenous chemical, physical and biological agents - genotoxins. These genotoxins alter its basic structural components - sugar residues, phosphodiester bonds, and nitrogenous bases. Organisms have therefore evolved a plethora of different strategies to both repair DNA lesions and maintain genomic stability. These DNA repair pathways are linked with several other cell pathways, including chromatin remodelling, DNA replication, transcription, cell cycle control, apoptosis - programmed cell death (PCD), thereby providing a coordinated cellular response to DNA damage. Biochemical mechanisms of DNA repair are relatively well understood in yeast and mammals, however, far less so in plants. While these repair mechanisms are evolutionary conserved, significant differences still remain. Therefore, further investigation is required. This thesis summarises the introduction of a novel plant model - the moss, Physcomitrella patens (Physcomitrella). As a haploid gametophyte with unique characteristics of high frequency of homologous recombination (HR), and apical growth of filaments, it is an ideal organism to study DNA repair in plants. Previous research on Physcomitrella regarding mechanisms of DNA lesion repair induced by...

Homologous recombination (HR) repair is critical for the maintenance of genomic stability, as it is involved in the precise repair of DNA double-strand breaks (DSBs) using an intact homologous template for repair. The initiation of 5' strand resection of DNA ends is a critical determinant in this process, which commits cells to HR repair and prevents repair by non-homologous end joining (NHEJ). The human single-stranded DNA (ssDNA) binding complexes, RPA and SOSS1, are involved in regulating DSB signaling and HR repair. In this study, I demonstrate a novel function of SOSS1 in HR repair, in which SOSS1 stimulates hExo1-dependent resection. Despite its poor activity in binding duplex DNA, SOSS1 facilitates hExo1 recruitment to duplex DNA ends and promotes its activity in resection independently of MRN in vitro. MRN(X) is a highly conserved complex that is involved in the early steps of HR repair by regulating DSB resection. MRN interacts with CtIP and constitutes resection machinery that can perform limiting processing on DNA ends. In this study, I also examine the biochemical activities of MRN and CtIP in DSB resection through reconstituted in vitro assays. I show that the ATP-dependent DNA unwinding activity of MRN is responsible for overcoming Ku inhibition of hExo1- and Dna2/BLM-dependent resection activity in vitro. I propose that this unwinding step displaces Ku away from the DNA ends and facilitates the recruitment of hExo1 to the DNA ends for efficient resection. In addition, I show that CtIP can promote overcoming the inhibitory effect of Ku in resection together with MRN. Further, I demonstrate that MRN nuclease activity is required for efficient processing of covalent adducts from DNA ends in vitro, suggesting that the nucleolytic removal of covalent adducts by MRN generates free ends for hExo1- and Dna2/BLM binding. Overall, this study provides mechanistic insight into the regulation of DSB resection in human cells. / text

ATM is the defective kinase in the neurodegenerative disorder ataxia telangiectasia. This kinase is associated with DNA double-strand break (DSB) repair and cell cycle control. Our laboratory previously demonstrated elevated levels of deletions and error-prone double-strand break repair via microhomology-mediated end joining (MMEJ) in ATM-deficient (A-T) extracts when compared to controls (wtATM+). To assess the involvement of enhanced nuclease activities in A-T extracts we studied the stability of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in detection from full-length products to shorter products in A-T extracts. Addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was dependent on its kinase activities. These results demonstrated a role for ATM in suppressing the degradation of DNA ends possibly through inhibiting nucleases implicated in MMEJ such as Mre11. Therefore, we assessed DNA end-stability in Mre11-depleted nuclear extracts and in extracts treated with the Mre11 nuclease inhibitor, Mirin. This resulted in decreased DNA degradation in both control and A-T extracts. Knockdown of Mre11 levels also led to an enhancement of DNA end-stability in nuclear extracts. Examining MMEJ levels by employing an in vivo reporter assay system revealed a decline in this pathway in Mre11-knockdown cells and in those treated with Mirin. These results signify a role for the Mre11 nuclease in MMEJ in mammalian cells and indicate a regulatory function for ATM in the control of error-prone DSB repair and preservation of DNA end-stability at a break.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-13T14:09:49Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Zenaide S Monte - Tese Doutorado - Defesa 15-jul-2016.pdf: 10509605 bytes, checksum: 9a625f96607c0ccae6d58b896ba38772 (MD5) / Made available in DSpace on 2017-07-13T14:09:49Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Zenaide S Monte - Tese Doutorado - Defesa 15-jul-2016.pdf: 10509605 bytes, checksum: 9a625f96607c0ccae6d58b896ba38772 (MD5) Previous issue date: 2016-07-15 / CAPES / Compostos heterocíclicos do tipo pirimidina são conhecidos pela enorme riqueza de seus potenciais biológicos e farmacológicos. Numerosas reações de heterociclização que permitem a obtenção dessa classe de compostos são igualmente reconhecidas, devido à diversidade de atividades e do conhecimento prévio de inúmeras viabilidades sintéticas. As modificações estruturais obtidas na busca da otimização das rotas sintéticas conferem às novas moléculas diferentes propriedades físicas e alteram a reatividade das moléculas, acarretando em mudanças na distribuição nas células e nos tecidos. Essas discretas modificações estruturais podem revelar efeitos biológicos que haviam estado latentes ou eclipsados pelos efeitos colaterais do composto protótipo. Sendo assim, o presente trabalho apresenta a síntese por método convencional dos derivados da pirimidina 3a-q e 5o-q. Estes compostos foram avaliados quanto às propriedades duais: Antimicrobiana e Fotoluminescência, estas apresentaram resultados promissores, podem atuar como boas sondas fluorescentes. Foram realizados também estudos Espectrofotométricos em diferentes solventes além de determinarmos o Cálculo do Orbital Molecular de Fronteira nesta série. Realizou-se também a nanoencapsulação em lipossomas para o composto 3o. Obtivemos também p-arilamidinas 3a-j por método convencional e avaliamos as propriedades Antimicrobiana e Citotóxica, as quais apresentaram resultados significativos. Realizamos a síntese dos derivados pirimidínicos 3a-f e 6a-f pelo método em Irradiação em micro-ondas, em fase sólida e em solução, respectivamente. Estudamos também para estes as propriedades Antimicrobiana e Antioxidante, cujos os resultados foram satisfatórios. Foram obtidos bons rendimentos para todos os compostos obtidos. Um estudo inédito de RMN bidimensional, COSY, HSQC e HMBC foram aplicados às pirimidinas foi realizado, proporcionando a caracterização estrutural destes compostos. Os resultados obtidos apresentaram significativa potencialidades biológicas, farmacológicas e fotofísicas. / Heterocyclic compounds of pyrimidine are known for rich biological and pharmacological potential. A number of heterociclic reactions that enable the obtention of this class of compounds are also recognized, due to the diversity of activities and prior knowledge of their various synthetic viabilities. The structural modifications obtained in search for the best synthetic routes confered different physical properties on the new molecules and altered the reactivity of the molecules, resulting in changes in the distribution in cells and tissues. These discrete structural changes may reveal biological effects that may have been latent or eclipsed by the side effects of the compound prototype. This work presents the synthesis by conventional method of derivatives from pyrimidine 3a-q and 5o-q. These compounds were evaluated for dual properties: antimicrobial and photoluminescence. They showed promising results can act as fluorescent probes. Spectroscopic studies were carried out with different solvents and the Frontier Molecular Orbital calculation for this series was determined. Nanoencapsulation of liposomes for the compound 3o was prepared. The conventional method was used to obtain 3a-j p-arilamidinas. Results were significant for the antimicrobial and cytotoxic properties. Finally, the synthesis of pyrimidines derivatives 3a-f and 6a-f by a new method, solid phase micro-wave and in solution, respectively was carried out. We have studied both the Antimicrobial and Antioxidant properties with satisfactory results. Good yields were obtained for all compounds obtained. New studies of two-dimensional NMR, COSY, HSQC and HMBC were performed besides the characterization of the compounds by standard methods. The results show that the compounds obtained have great biological, pharmacological potential and Photophysical.

No description available.

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2007. / Title from second page of PDF document. Includes bibliographical references (p. 91-102).

The presence of introns in all the eukaryotic genomes identified so far underscores the fundamental and ubiquitous role of pre-mRNA splicing. The spliceosomal machinery, comprised of five small nuclear RNAs and several protein factors, catalyzes the two-transesterification reactions of splicing with precision and consistency. Through a complex network of protein-protein and RNA-protein interactions it ensures the removal of the intron and ligation of the flanking exons to yield the mature mRNA. Prpl7 is a splicing factor that functions at the second-step of splicing (Vijayraghavan et all, 1989). Null alleles of prpl7 are viable at 23°C but die at temperatures above 33°C (Jones et al.9 1995). Besides its functions in pre-mRNA splicing, mutants in PRP17ICDC40 were independently shown to affect cell-cycle progression, particularly the Gl/S and G2/M transitions (Chawla et a/., 2003). In this study, we have attempted a further characterization of Prpl7 to analyze both its role in pre-mRNA splicing and in cell-cycle progression with an aim to decipher underlying reasons for the interlinking of these two cellular processes. Different experimental approaches were adopted to achieve this goal. Global gene-expression profiling provided an overview of all the transcripts affected in a prpl 7 mutant and allowed its comparison with mutants of other splicing factors. This exercise aided in identification of both pre-mRNA splicing and cell-cycle related effects of Prpl7. Biochemical analysis of the Prpl7 spliceosomal associations have provided further clarity on the part played by Prpl7 in pre-mRNA splicing. A genome-wide two-Hybrid screen for interacting partners of Prpl7 was undertaken and uncovered two Likely interacting partners of Prpl7. Global expression profiling of splicing mutants Pleiotropic phenotypes observed in mutants of prpl 7 and few other splicing factors have been speculated to arise from either the multi functionality of the factor or more likely due to a specific requirement of the factor in splicing of a select subset of transcripts, that encode proteins essential to the affected cellular pathway. These observations raise questions about the ubiquitous requirement of factors in pre-mRNA splicing. To understand these aspects of splicing, we studied the effects of splicing factor mutants on a genome-wide scale. Using splicing-sensitive DNA microarrays imprinted with all yeast ORFs and in addition, independent spots for a majority of the intron sequences, we analyzed the global expression changes triggered by the inactivation of temperature-sensitive mutations in PRP17 or PRP22. Experiments with prp2-l mutant strain detect, as expected, an increase in pre-mRNA levels at the intron spots and further demonstrated that the ORF spots detect a decrease in mRNA levels in these DNA microarrays. These results established the DNA micro arrays as tools for the analysis of splicing on a global scale. The temporal alterations in transcript profiles in prpl 7 and prp22 mutants, as compared to the wild type, revealed both shared and unique effects of these factors on clusters of intron-containing transcripts. Such differential effects, on intron-containing transcripts, amongst the splicing mutants implicate specialized roles for each of these factors. Through analysis of the set of intron-containing transcripts affected in prpl7Δ cells, we infer those attributes of these pre-mRNA substrates, which predispose a need for Prpl 7. We find that splicing of introns longer than 200nts has a stronger dependence on Prpl7. The distance between consensus intron elements- the branch-nucleotide and the 3'splice-site (B), also imposes a requirement for Prpl7. Introns with a 13nts or lesser distance between these elements are spliced even in the absence of Prpl 7, both in vivo and in vitro. The 5'splice-site to branch-nucleotide distance (A) also influences the need for Prpl7. Most introns with a A/B ratio of less than 2 undergo Prpl7 independent splicing in vivo. Intron-containing genes that could be responsible for the pleiotropic phenotypes of prpl7 were also identified through the global splicing analysis. These included splicing targets that act at the Gl-S phase such as ANC1/TAF14, TMD4, PHO85 and those at the G2-M phase of the cell-cycle; TUB], TUB3, GIM5, MOBl UBC9. Recently, a different study implicates ANC1ITAF14 as the intron-containing gene responsible for the cell-cycle phenotype associated with prpl7 (Dahan and Kupiec, 2004). Our global analysis of all intron-containing transcripts with compromised expression in prpl7A cells identify, in addition, PHO85 as a possible regulator underlying cell-cycle effects in this mutant. Pho85 is a cyclin-dependent kinase that functions at both the Gl/S and M/Gl phases of the division cycle (Moffate* al., 2000). Synergistic growth defects in double mutants of prpl7 and pho85 have uncovered a novel role for Prpl7 in bud morphogenesis. Our micro array data also reveals compromised expression levels for several key intronless cell-cycle rregulatory genes indicating a possible splicing-independent role for Prpl7 in the cell-cycle. Examples of such transcripts are: the Gl cyclins CLN1, CLN2 and CLN3; CDC6, required for assembly of the pre-replication complex at sites of replication origin; and the cell-cycle regulatory transcription factors: SWI5 and ACE2. The global analysis has therefore enabled, for the first time, a characterization of the splicing substrate specificity of Prpl7 and has also uncovered the effects of this protein on gene expression during cell-cycle progression (Fig. V.I A). Spliceosomal interactions of Prpl7 To understand the function and associations of Prpl7 in the spliceosome, we have examined its snRNP interactions and determined the time point of its coalescence on assembling spliceosomes. A functional epitope tagged-Prpl7 was created using the polyoma middle T-antigen and the poly-HIS tags (Stevens et aln 1999). Through immunoprecipitation analyses performed with splicing extracts, from this strain, we find Prpl7 to associate with three spliceosomal snRNPs- U2, U5 and U6, implicating an interaction with active spliceosomes or post-splicing complexes. Specific biochemical depletion of any one of these snRNAs, through oligo-directed RNaseH cleavage, did not have a drastic effect on the association of Prpl7 with the other two snRNAs. To decipher the point at which Prp 17 joins the assembling spliceosomes, we examined the presence of Prp 17 in in vitro assembled complexes generated under various conditions. The conditions adopted were designed to stall and enrich for •assembly intermediates. A co-immunoprecipitation of the input precursor RNA and reaction intermediates revealed an early association of Prp 17 with the assembling Spliceosome prior to its catalytic activation. This association occurred in the A2-1 complex, which contains the U4/U6.U5 tri-snRNP along with the Ul and U2snRNPs. Prpl7 was found to associate with all subsequent complexes until the completion of catalytic transesterification reactions and possibly continue with the spliced-out introns complex (Fig. V.1B). Identification of two novel interacting partners of Prpl7 from a genome-wide two-hybrid screen Although several genetic interacting partners of PRP17 are known, none display a direct physical association with Prpl7. Knowledge of the proteins that Prpl7 interacts with can further the functional characterization of this protein and aid in deciphering its link to cell-cycle progression. A genome-wide screen for interacting partners using Prpl7 as bait was carried out in a two-hybrid system with a yeast genomic DNA-B42 activation-domain library (Gyuris et al., 1993). Through this screen we identified two interacting partners of Prpl 7- YOL078W, an essential gene and SGML The domain in the 1176 amino acid YOL078W protein responsible for interaction with Prpl7 was mapped to a 225 amino acid segment in the C-terminai region of this protein. The N-terminal region of the protein appears to exert a negative effect on the interaction with Prpl7. While YOL078w does not have any apparent role in pre-mRNA splicing, a majority of the cells arrest with small buds indicating a late Gl or early S phase arrest upon transcriptional shut-down of YOL078W. YOL078W has been independently characterized as AVOl, a component of the TOR complex, involved in nutrient sensing and cell size regulation (Loewith et al, 2002). Other reports show it tto be a component of a complex that interacts with Ceglp, a nuclear protein involved in mRNA capping (Gavin et al, 2002). We hypothesize that Prpl7 and Avol may exist in a dynamic nucleocytoplasmic complex possibly functioning in either cell-cycle regulation, RNA processing or both. Through this study we have Established the use of splicing-sensitive microarrays as tools for the characterization of pre-mRNA splicing factors. Simultaneous assessment of the effects on other cellular pathways was accomplished through expression profiling of all the intron-containing and intronless genes. Deciphered the differential dependence of pre-mRNA substrates on spliceosome factors at a global scale. Predicted the substrate-specificity of the second-step splicing factor, Prpl7, and verified some of these predictions in vitro. Gathered evidence for a possible splicing-independent effect of Prpl7 on the cell division cycle. Uncovered a novel function of Prpl7 in bud morphogenesis, as deduced from its synergistic genetic interaction with PHO85. Identified U2, U5 and U6 snRNPs as interacting partners of Prpl7 in both xtracts and in in vitro splicing reactions. Determined the point of coalescence of Prpl7 during spliceosome assembly to be at an early assembly stage soon after the entry of U4/U6.U5 tri-snRNP and prior to catalytic activation. Demonstrated continued Prpl7 association with the spliceosome beyond the completion of the splicing reactions. Identified Avolp and Sgmlp as novel interacting partners of Prpl7 through a genome-wide two-hybrid screen.

61 Summary Wilms' tumor gene 1 (WT1) is highly expressed in acute leukemia and other hematological malignancies. It has been therefore suggested as a potential universal marker of minimal residual disease (MRD), particularly in patients with acute myeloid leukemia (AML). Due to controversial results of some of the studies, the role of WT1 in MRD follow-up and WT1 prognostic significance remain unclear. WT1 protein is produced in more than 36 different isoforms. These variants have distinct, partially overlapping functions and their ratio is supposed to influence the final effect of WT1. However, despite the increasing number of studies, the clinical impact of WT1 and its isoforms in acute leukemia have not yet been elucidated. We established a unique qPCR method to assess the expression pattern of the main 4 WT1 isoforms. Using this method, we determined the ratio of WT1 variants in the samples of patients with AML, myelodysplastic syndrome (MDS) and healthy controls. Our data showed that this pattern can distinguish among particular hematological malignancies, but lacks a prognostic significance. Within our international study group we determined the prognostic significance of total WT1 expression in childhood AML. Based on our results of a large cohort of patients we can conclude that WT1 expression at...