Transcriptome analysis of the mobile genome ICEclc in Pseudomonas knackmussii B13.

Background: Integrative and conjugative elements (ICE) form a diverse group of DNA elements that are integrated in the chromosome of the bacterial host, but can occasionally excise and horizontally transfer to a new host cell. ICE come in different families, typically with a conserved core for functions controlling the element's behavior and a variable region providing auxiliary functions to the host. The ICEclc element of Pseudomonas knackmussii strain B13 is representative for a large family of chromosomal islands detected by genome sequencing approaches. It provides the host with the capacity to degrade chloroaromatics and 2-aminophenol. Results: Here we study the transcriptional organization of the ICEclc core region. By northern hybridizations, reverse-transcriptase polymerase chain reaction (RT-PCR) and Rapid Amplification of cDNA Ends (5'-RACE) fifteen transcripts were mapped in the core region. The occurrence and location of those transcripts were further confirmed by hybridizing labeled cDNA to a semi-tiling micro-array probing both strands of the ICEclc core region. Dot blot and semi-tiling array hybridizations demonstrated most of the core transcripts to be upregulated during stationary phase on 3-chlorobenzoate, but not on succinate or glucose. Conclusions: The transcription analysis of the ICEclc core region provides detailed insights in the mode of regulatory organization and will help to further understand the complex mode of behavior of this class of mobile elements. We conclude that ICEclc core transcription is concerted at a global level, more reminiscent of a phage program than of plasmid conjugation.

Biochemical characterization of recombinant human telomerase

Résumé Les télomères sont les structures ADN-protéines des extrémités des chromosomes des eucaryotes. L'ADN télomérique est constitué de courtes séquences répétitives. L'intégrité des télomères est essentielle pour protéger les extrémités des chromosomes contre les systèmes de dégradations et pour les distinguer des cassures de l'ADN double brin. Parce que la machinerie de la réplication de l'ADN n'est pas capable de répliquer l'extrémité des chromosomes, les télomères raccourcissent au fur et à mesure des cycles de réplication. Dès que les télomères atteignent une longueur critique, leur structure protectrice est perdue. Cela induit un signal de dommage de l'ADN et l'arrêt du cycle cellulaire. Pour contrebalancer le raccourcissement des télomères, les cellules qui s'auto régénèrent, dont les cellules de la moelle osseuse, les lymphocytes activés et 80-90% des cellules cancéreuses, expriment la télomérase. C'est une ribonucléoprotéine qui a la capacité de synthétiser des séquences télomériques par transcription inverse d'une courte séquence contenue dans sa propre sous-unité ARN avec laquelle elle est associée. La télomérase humaine est une enzyme processive au niveau de l'addition des nucléotides et aussi des répétitions télomériques. La télomérase de levure et la télomérase humaine sont toutes deux dimériques et il a été montré que la télomérase humaine recombinante contient deux ARN qui coopèrent pour fonctionner ainsi que deux sous-unités catalytiques. Cependant, il n'a pas encore été montré quel est le rôle de la dimérisation dans l'activité de la télomérase. Afin d'élucider ce rôle, nous avons exprimé, reconstitué et purifié la télomérase humaine dimérique recombinante. Et pour étudier l'effet d'ARN mutants sur l'activité de la télomérase, nous avons développé une méthode pour reconstituer et enrichir en hétérodimères de télomérase. Les hétérodimères contiennent une sous-unité ARN sauvage et une sous-unité ARN mutée au niveau de la séquence de la matrice. Sur l'ARN muté nous avons introduit une étiquette aptamer ARN-S1 puis nous avons purifié la télomérase via l'etiquette Si. Nous avons montré que la dimérisation est essentielle pour l'activité de la télomérase. Nos données indiquent que chaque télomérase du dimère allonge leur substrat, l'ADN télomérique, indépendamment l'une de l'autre à chaque cycle d'élongation mais que l'addition itérative de répétitions télomériques nécessite une coopération entre les deux télomérases du dimère. Nous proposons donc un modèle dans lequel les deux télomérases du dimères se lient et allongent deux substrats télomères et que pendant l'élongation processive les deux enzymes subissent un changement de conformation de manière coordonnée, ce changement va permettre le repositionnement des substrats pour d'autres cycles d'additions de répétitions télomériques. Dyskeratosis congenita est une maladie mortelle due majoritairement au disfonctionnement de la moelle osseuse. Dans la forme autosomale de la maladie, l'ARN de la télomérase contient des mutations. En utilisant notre système de reconstitution, nous avons montré que ces ARN mutés, qui ont perdu leur activité enzymatique dans le cas d'un homodimère de mutants, sont dominant négatifs quand ils sont présents dans les hétérodimères sauvage/mutant. Cet effet trans-dominant négatif pourrait contribuer à la progression de la maladie. Abstract Telomeres are protein-DNA structures at the ends of linear eukaryotic chromosomes. The telomeric DNA consists of tandemly repeated sequences. Telomeric integrity is essential to protect chromosomal ends from nucleolytic degradation and to prevent their recognition as DNA double strand breaks. Due to the inability of the conventional DNA replication machinery to replicate terminal DNA stretches, telomeres shorten with continuous rounds of DNA replication. As soon as telomeres reach a critical length, their protective structure is lost and the deprotected telomeres will induce a DNA damage response leading to cell cycle arrest. To counteract telomere shortening, self-renewing cells, including bone marrow cells, activated lymphocytes and 80-90% of cancer cells express the cellular reverse transcriptase telomerase, which has the capacity to synthesize telomeric repeats by reverse transcription of a short template sequence encoded by its stably associated RNA subunit. Human telomerase is a processive enzyme for nucleotide as well as repeat addition. Both yeast and human telomerase are dimeric enzymes and recombinant human telomerase has been shown to contain two functionally cooperating RNAs and most probably also two protein subunits. However, it has remained unclear how dimerization may contribute to telomerase activity. To study the role of dimerization, we expressed, reconstituted and purified recombinant human telomerase. We also developed a new method to reconstitute and enrich for telomerase heterodimers containing wild-type (wt) and mutant telomerase RNA subunits. To this end we introduced an S1-RNA-aptamer tag into telomerase RNA and purified telomerase reconstituted with a mixture of untagged and tagged RNA via the S1-tag. Using this experimental system, we introduced template mutations in the tagged RNA subunit and examined the effect of mutant RNAs on wt telomerase activity in wt/mutant heterodimers. We obtained evidence that dimerization is essential for telomerase activity. Our data indicate that the two subunits elongate telomere substrates independently of each other during single rounds of elongation, but that iterative addition of telomeric repeats requires cooperation between the two subunits. We suggest a model, in which dimeric telomerases bind and elongate two telomere substrates and that the two subunits undergo coordinated conformational changes during processive elongation that enable repositioning the substrates for subsequent rounds of repeat addition. Dyskeratosis congenita is a multisystemic disease with bone marrow failure as the major cause of death. The autosomal form of this disease was found to harbor mutations in the telomerase RNA. Using our reconstitution system, we tested whether mutant dyskeratosis telomerase RNAs behaved in a dominant negative manner. We observed that dyskeratosis telomerase RNA mutants, which lacked enzymatic activity were dominant negative, when present in wt/ mutant heterodimers. The transdominant negative effect of these mutants may contribute to disease progression.

Analysis of a boundary protein delimiting chromatin domains in yeast

SUMMARY The expression state of a eukaryotic gene depends in part on its location in the chromosome. This position effect results from the organization of eukaryotic genomes into discrete functional domains, defined by local differences in chromatin structure. The expression of genes within each domain appears to be defined and maintained by the concerted action of regulatory elements such as promoters, enhancers, silencers and locus control regions. Individual domains may be bordered by boundary elements that separate regions of permissive and silent chromatin. When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres towards more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one of these transcription factors, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino-acid substitutions that similarly inhibited the boundary and histone-binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains. RESUME L'expression des gènes eucaryotes dépend en partie de leur localisation sur les chromosomes. Cet effet de position résulte de l'organisation des génomes eucaryotes en domaines fonctionnels, définis par des changements locaux au niveau de la structure de la chromatine. Dans chacun de ces domaines, l'expression des gènes est définie et maintenue par l'action concertée de différents éléments régulateurs tels que les promoteurs, les amplificateurs, les silenceurs et les locus control régions. Ces domaines peuvent être entourés par des éléments barrière, séparant les régions de chromatine répressive des régions permissive pour l'expression des gènes. Lorsqu'ils se situent à proximité d'éléments chromosomiques comme les telomères, les gènes peuvent être réprimés de manière épigénétique. Chez la levure, cette répression est établie par la propagation des protéines SIR depuis les télomères vers les régions centromériques. Certains facteurs de transcription peuvent empêcher la répression d'un gène, lorsqu'ils sont placés entre ce gène et le télomère. Nous avons étudié un de ces facteurs, CTF-1, qui a la particularité de lier directement l'histone H3. La délétion de certaines parties de CTF-1 a permis de déterminer que la région responsable de l'activité barrière correspond au domaine d'interaction avec H3. Plusieurs mutations points effectuées dans ce domaine inhibent à la fois l'activité barrière et la capacité de lier H3. Des expériences d'immuno-précipitation de la chromatine indiquent que la protéine barrière CTF-1 prévient efficacement la propagation des protéines SIR et sépare des domaines contenant des histones hypo-acétylées de ceux constitués d'histones hyper-acétylées. Ces résultats suggèrent que CTF-1 interagit directement avec l'histone H3 pour empêcher la propagation de la chromatine répressive, délimitant ainsi des domaines de chromatine permissive et des domaines de chromatine silencieuse.

Long time behaviour of diffusing particles in constrained geometries; application to chromatin motion

Inspired by experiments that use single-particle tracking to measure the regions of confinement of selected chromosomal regions within cell nuclei, we have developed an analytical approach that takes into account various possible positions and shapes of the confinement regions. We show, in particular, that confinement of a particle into a subregion that is entirely enclosed within a spherical volume can lead to a higher limit of the mean radial square displacement value than the one associated with a particle that can explore the entire spherical volume. Finally, we apply the theory to analyse the motion of extrachromosomal chromatin rings within nuclei of living yeast.

Current cytogenetic map of the common shrew, Sorex araneus L.: localization of 7 genes and 4 microsatellites

Here we present information on the assignment of 7 genes, ACADVL, ADORA3, ATP7A, MTMR4, MYH2, HBB, TSPAN-3, and 4 common shrew microsatellites to chromosomes of the common shrew (Sorex araneus) and on the current status of its cytogenetic map. Comparative mapping data were used for the analysis of evolutionary chromosomal rearrangements in the common shrew genome.

Molecular genetics of rare hereditary diseases via genome-wide and integrated approaches

ABSTRACTThe Online Mendelian Inheritance in Man database (OMIM) reports about 3000 Mendelian diseases of known causal gene and about 2000 that remain to be mapped. These cases are often difficult to solve because of the rareness of the disease, the structure of the family (too big or too small) or the heterogeneity of the phenotype. The goal of this thesis is to explore the current genetic tools, before the advent of ultra high throughput sequencing, and integrate them in the attempt to map the genes behind the four studied cases. In this framework we have studied a small family with a recessive disease, a modifier gene for the penetrance of a dominant mutation, a large extended family with a cardiac phenotype and clinical and/or allelic heterogeneity and we have molecularly analyzed a balanced chromosomal translocation.RESUMELa base de données des maladies à transmission mendélienne, Online Mendelian Inheritance in Man (OMIM), contient environ 3000 affections à caractère mendélien pour lesquelles le gène responsable est connu et environ 2000 qui restent à élucider.Les cas restant à résoudre sont souvent difficiles soit par le caractère intrinsèquement rare de ces maladies soit à cause de difficultés structurelles (famille trop petite ou trop étendue) ou hétérogénéité du phénotype ou génétique. Cette thèse s'inscrit avant l'arrivée des nouveaux outils de séquençage à haut débit. Son but est d'explorer les outils génétiques actuels, et de les intégrer pour trouver les gènes impliqués dans quatre cas représentant chacun une situation génétique différente : nous avons étudié une famille de quatre individus avec une transmission récessive, recherché un gène modificateur de la pénétrance de mutations dominantes, étudié une famille étendue présentant un phénotype cardiaque cliniquement et/ou allèliquement hétérogène et nous avons fait l'analyse moléculaire d'une translocation chromosomique balancée.

Automated four-color interphase fluorescence in situ hybridization approach for the simultaneous detection of specific aneuploidies of diagnostic and prognostic significance in high hyperdiploid acute lymphoblastic leukemia.

In high hyperdiploid acute lymphoblastic leukemia (ALL), the concurrence of specific trisomies confers a more favorable outcome than hyperdiploidy alone. Interphase fluorescence in situ hybridization (FISH) complements conventional cytogenetics (CC) through its sensitivity and ability to detect chromosome aberrations in nondividing cells. To overcome the limits of manual I-FISH, we developed an automated four-color I-FISH approach and assessed its ability to detect concurrent aneuploidies in ALL. I-FISH was performed using centromeric probes for chromosomes 4, 6, 10, and 17. Parameters established for nucleus selection and signal detection were evaluated. Cutoff values were determined. Combinations of aneuploidies were considered relevant when each aneuploidy was individually significant. Results obtained in 10 patient samples were compared with those obtained with CC. Various combinations of aneuploidies were identified. All clones detected by CC were observed also by I-FISH, and I-FISH revealed numerous additional abnormal clones in all patients, ranging from < or =1% to 31.6% of cells analyzed. We conclude that four-color automated I-FISH permits the identification of concurrent aneuploidies of potential prognostic significance. Large numbers of cells can be analyzed rapidly. The large number of nuclei scored revealed a high level of chromosome variability both at diagnosis and relapse, the prognostic significance of which is of considerable clinical interest and merits further evaluation.

Clinical implications of molecular neuropathology and biomarkers for malignant glioma.

Malignant gliomas are currently diagnosed based on morphological criteria and graded according to the World Health Organization classification of primary brain tumors. This algorithm of diagnosis and classification provides clinicians with an estimated prognosis of the natural course of the disease. It does not reflect the expected response to specific treatments beyond surgery (eg, radiotherapy or alkylating chemotherapy). Clinical experience has revealed that gliomas sharing similar histomorphological criteria might indeed have different clinical courses and exhibit highly heterogenous responses to treatments. This was very impressively demonstrated first for oligodendrogliomas. The presence or lack of combined deletions of the chromosomal segments 1p/19q was associated with different benefit from radiotherapy and chemotherapy. We review current molecular markers for malignant gliomas and discuss their current and future impact on clinical neuro-oncology.

Prenatal diagnosis and epidemiology of multicystic kidney dysplasia in Europe.

OBJECTIVES: The aim of this study is to describe the prenatal diagnosis and epidemiology of multicystic kidney dysplasia (MCKD). METHODS: The study is based on routinely collected data from a European database of major congenital anomalies including 13 registries with cases born in 1997-2006 and covering 1 458 552 births. RESULTS: There were 601 MCKD cases giving an overall prevalence of 4.12 per 10 000 births with regional variation. In live births, 87% of cases had an isolated renal anomaly and 13% had associated major nonrenal anomalies (chromosomal, syndrome or other major anomalies). For the cases with isolated renal anomalies, 51/386 (11%) and 7/386 (2%) choose to terminate the pregnancy or resulted in an intrauterine fetal death, respectively. The prenatal detection rate was 88% in both unilateral and bilateral cases. Birth outcome differed with 92% of unilateral MCKD cases being liveborn compared with 33% of bilateral MCKD cases. For unilateral MCKD cases, 84% had an isolated renal anomaly compared with 51% of bilateral MCKD cases (p < 0.001). CONCLUSIONS: Cases with unilateral MCKD are mainly liveborn, and only 16% have associated major malformations or a syndrome. Cases with bilateral MCKD are often associated with nonrenal major congenital anomalies or part of a syndrome, and only one third of bilateral MCKD cases in this study were liveborn. Prenatal detection rate of MCKD was high for both unilateral and bilateral cases. © 2014 John Wiley & Sons, Ltd.

Evaluation of the prenatal diagnosis of limb reduction deficiencies. EUROSCAN Study Group.

Ultrasound scans in the mid-trimester of pregnancy are now a routine part of antenatal care in most European countries. Using data from registries of congenital anomalies a study was undertaken in Europe. The objective of the study was to evaluate prenatal detection of limb reduction deficiencies (LRD) by routine ultrasonographic examination of the fetus. All LRDs suspected prenatally and all LRDs (including chromosome anomalies) confirmed at birth were identified from 20 Congenital Malformation Registers from the following 12 European countries: Austria, Croatia, Denmark, France, Germany, Italy, Lithuania, Spain, Switzerland, The Netherlands, UK and Ukrainia. These registries are following the same methodology. During the study period (1996-98) there were 709,030 births, and 7,758 cases with congenital malformations including LRDs. If more than one LRD was present the case was coded as complex LRD; 250 cases of LRDs with 63 (25.2%) termination of pregnancies were identified including 138 cases with isolated LRD, 112 with associated malformations, 16 with chromosomal anomalies and 38 non chromosomal recognized syndromes. The prenatal detection rate of isolated LRD was 24.6% (34 out of 138 cases) compared with 49.1% for associated malformations (55 out of 112; p<0.01). The prenatal detection of isolated terminal transverse LRD was 22.7% (22 out of 97), 50% (3 out of 6) for proximal intercalary LRD, 8.3% (1 out of 12) for longitudinal LRD and 0 for split hand/foot; for multipli-malformed children with LRD those percentages were 46.1% (30 out of 65), 66.6% (6 out of 9), 57.1% (8 out of 14) and 0 (0 out of 2), respectively. The prenatal detection rate of LRDs varied in relation with the ultrasound screening policies from 20.0% to 64.0% in countries with at least one routine fetal scan.

Excision of the Staphylococcal Cassette Chromosome mec (SCCmec) and its crucial role in the horizontal transfer of methicillin resistance in staphylococci.

Staphylococcus aureus est un pathogène humain majeur ayant développé des résistances contre la quasi totalité des antibiotiques disponibles, incluant la très importante famille des β- lactamines. La résistance à cette classe d'antibiotiques est conférée par la « Staphylococcal Cassette Chromosome mec » (SCCmec), qui est un élément génétique mobile capable de s'insérer dans le chromosome bactérien et capable d'être transféré horizontalement chez d'autres staphylocoques. Le mécanisme moléculaire impliqué dans ce transfert horizontal demeure largement inconnu. L'une des premières étapes du transfert est l'excision du SCC mec du chromosome bactérien. Cette excision est promue par des enzymes codées par l'élément SCCmec lui- même et appelées de ce fait « Cassette Chromosome Recombinases » (Ccr). L'un des buts de ce travail de thèse a été de comprendre la régulation de l'expression des gènes codant pour les Ccr recombinases. En utilisant des outils moléculaires originaux, nous avons été en mesure de démontrer en premier lieu que les Ccr recombinases étaient exprimées de façon « bistable », c'est à dire qu'uniquement quelques pourcents de cellules dans une population exprimaient ces gènes à un temps donné. Dans un deuxième temps, nous avons également démontré que l'expression de ces gènes était régulée par des facteurs étrangers au SCC mec. L'expression bistable des recombinases est un concept important. Effectivement, cela permet à la majorité des cellules d'une population de conserver l'élément SCC mec, alors que seulement une petite fraction le perd afin de le rendre disponible pour un transfert. Ainsi, alors que l'élément SCC mec continue de se propager avec la multiplication des bactéries Staphylococcus aureus résistant à la méticilline (SARM), il peut être simultanément transmis à des souches susceptibles (Staphylococcus aureus susceptible à la méticilline, SASM), entraînant l'apparition de nouveaux SARM. De façon très intéressante, le fait que cette bistabilité est contrôlée par les bactéries, et non le SCCmec lui-même, montre que la décision de transférer ou non la cassette SCC mec appartient à la bactérie. En conséquence, il doit exister dans la nature des souches qui sont plus ou moins aptes à effectuer ce transfert. En nous appuyant sur ces observations, nous avons montré que l'excision du SCC mec était effectivement régulée de façon très étroite au cours de la division cellulaire, et ne se passait que pendant un temps limité au début de la croissance. Ce résultat est compatible avec une régulation génétique commandée par la densité cellulaire, qui pourrait être dépendante de la production de signaux extracellulaires, du type que l'on rencontre dans le quorum sensing. Les signaux hypothétiques entraînant l'excision du SCC mec restent inconnus à l'heure actuelle. La connaissance de ces signaux pourrait se révéler très importante afin de développer des stratégies pour interférer avec la dissémination de la résistance au β-lactamines. Deux sujets additionnels ont été logiquement investigués au vu de ces premiers résultats. Premièrement, si certaines souches de SARM sont plus ou moins aptes à déclencher l'excision du SCC mec, de même certaines souches de SASM devraient être plus ou moins aptes à acquérir cet élément. Deuxièmement, afin d'étudier ces mécanismes de transfert au niveau épidémiologique, il nous a été nécessaire de développer des outils nous permettant d'explorer le phénomène à une plus large échelle. Concernant le premier point, il a été postulé que certains SASM seraient réfractaires à l'intégration génomique d'un SCC mec en raison de polymorphismes particuliers à proximité du site d'insertion chromosomique (attB). En étudiant plus de 40 isolais de S. aureus, provenant de porteurs sains, nous avons confirmé ce polymorphisme dans l'environnement à'attB. De plus, nous avons pu montrer que ces régions polymorphiques ont évolué parallèlement à des groupes phylogénétiques bien connus. Ainsi, si des telles régions réfractaires à l'intégration de SCC mec existent, celles-ci devraient ségréger dans des complexes clonaux bien définis qui devraient être facilement identifiables au niveau épidémiologique. Concernant le second point, nous avons été capables de construire un système rapporteur de l'excision du SCCmec, en utilisant un plasmide à faible copie. Ce système consistait en un promoteur fort et un gène codant pour une protéine verte fluorescente (GFP) sous le contrôle d'un promoteur fort séparés à l'aide d'un élément SCC artificiel portant trois terminateurs de transcription. Ainsi, la fluorescence ne s'exprime que si l'élément SCC est excisé du plasmide. Ce système a été testé avec succès dans plusieurs types de staphylocoques, et est actuellement évalué dans d'autres souches et conditions stimulant ou inhibant l'excision. De manière générale, cette dissertation représente parcours scientifique à travers plusieurs aspects d'un problème de santé publique majeur en rapport avec la résistance bactérienne aux antibiotiques. Ce travail s'attaque à des problèmes fondamentaux concernant le transfert horizontal de l'élément SCC mec. De plus, il s'intéresse à des aspects plus généraux de cet élément génétique mobile qui pourraient se révéler très importants en terme de mouvement de gènes au sein des staphylocoques, voir d'autres bactéries gram-positives. Finalement ce travail de thèse met en place le fondamentaux requis pour des recherches futures visant à interférer avec le transfert horizontal de la résistance aux β-lactamines. - Staphylococcus aureus is a major human pathogen. Moreover, S. aureus have developed resistance to almost all available antibiotics, including the important family of β-lactam molecules. Intrinsic resistance to β-lactams is conferred by the Staphylococcal Cassette Chromosome mec (SCCmec), which is a mobile genomic island that inserts into the staphylococcal chromosome and can be horizontally transferred into other staphylococci. However, little is known about the molecular mechanisms involved in this horizontal transfer into naïve strains. One of the first steps in SCC mec horizontal transfer is its excision from the chromosome. Excision is mediated by recombinase enzymes that are encoded by SCC mec itself, and named accordingly Ccr recombinases - for Cassette Chromosome recombinases. One goal of this thesis was to understand the regulation these recombinase genes. By using original molecular tools we could demonstrate first that the Ccr recombinases were expressed in a "bistable" manner, i.e. in only few percentages of the bacterial cells at a given time, and second that they were regulated by determinants that were not encoded on the SCC mec element, but elsewhere on the staphylococcal genome. "Bistable" expression Ccr recombinases is an important concept. It allows SCC mec to be excised and thus available for horizontal transfer, while ensuring that only some cells, but not the whole population, loose their valuable SCC mec genes. Thus, while the SCC mec element expands with the multiplication of the MRSA colony, it can simultaneously be transmitted into methicillin-susceptible S. aureus (MSSA), which convert into new MRSA. Most interestingly, the fact that bistability was regulated by the cells, rather than by SCC mec, indicates that it was the choice of the bacteria to trigger or not SCC mec transfer. As a consequence, there must be, in nature, staphylococcal strains that are more or less prone to sustain SCC mec transfer. Following these seminal observations we found that excision was indeed tightly regulated during bacterial division, and occurred only during a limited period of time at the beginning of bacterial growth. This is compatible with cell-density mediated gene regulation, and may depend on the production of extracellular signal molecules that transmit appropriate orders to neighboring cells, such as in quorum sensing. The potential signal triggering SCCmec excision is as yet unknown. However, it could be critical in promoting the horizontal transfer of methicillin resistance, or for the possible development of means to interfere with it. Two additional hypothesis were logically investigated in the view of these first results. First, if some strains of MRSA might be more prone than others to promote SCC mec excision, then some strains of MS SA might be more or less prone to acquire the element as well. Second, to investigate these multiple mechanisms at an epidemiological level, one would need to develop tools amenable to explore S. aureus strains at a larger scale. Regarding the first issue, it was postulated by others that some MSSA might be refractory to SCC mec integration because they had peculiar DNA polymorphisms in the vicinity of the site-specific chromosomal entry point {attB) of SCC mec. By studying >40 S. aureus isolates from healthy carriers, we confirmed the polymorphism of the attB environment. Moreover, we could show that these polymorphic regions co-evolved with well-known phylogenic clonal clusters. Therefore, if SCCwec-refractory attB environments exist, then they would segregate in well- defined S. aureus clonal clusters that would be easy to identify at the epidemiological level. Regarding the second issue, we were able to construct a new excision reporter system in a low copy number S. aureus plasmid. The reporter system consists in a strong promoter driving a green fluorescent protein {gfp) gene, separated by an artificial SCC-like element carrying three transcriptional terminators. Thus, fluorescence is not expressed unless the SCC-like element is excised. The system has been successfully tested in several aureus and non- aureus staphylococci, and is now being applied to more strains and various excision- triggering or inhibiting conditions. Altogether the dissertation is a scientific journey through various aspects of a salient medical problem with regard to antibiotic resistance and public health threat. The research work tackles fundamental issues about the mechanisms of horizontal transfer of the SCC mec element. Moreover, it also addresses more general features of this mobile element, which could be of larger importance with regard to gene trafficking in staphylococci, and maybe other gram-positive bacteria. Finally, the dissertation sets the fundamentals for future work and possible new ways to interfere with the horizontal transfer of methicillin resistance.

Epithelioid sarcoma: a clinicopathologic and immunohistochemical analysis of 106 cases from the French sarcoma group.

Epithelioid sarcoma (ES) is rare with a poor prognosis and for which a loss of INI1 expression has been recently reported. We report a study of 106 cases with clinical, histologic, and immunohistochemical data, including INI1 expression, and follow-up data. Of the 106 cases, 70 were the conventional subtype and 36 the large cell subtype. INI1 was negative in 86 cases (81.1%): 57 (81%) of 70 conventional and 29 (81%) of 36 large cell subtypes. Treatment modalities were available for 76 and follow-up for 80 patients. Of the 80 patients, 43 (54%) experienced metastasis and 25 (31%) died of the disease. Univariate analysis indicated that tumor size and mitotic index were significant for metastasis-free survival, whereas proximal location, tumor size, tumor multifocality, and mitotic index were significant for overall survival. Loss of expression of INI1 is frequent in the conventional and large cell subtypes of ES and can be used as a diagnostic marker, but it has no prognostic impact.

Chromatin domain boundaries delimited by a histone-binding protein in yeast.

When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres toward more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one such transcription factor, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to the CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino acid substitutions that similarly inhibited the boundary and histone binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains.