The Drosophila RAD21 cohesin persists at the centromere region in mitosis

‘Cohesin’ is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prophase, well before sister chromatids separate (reviewed in [1 and 2]). Here we address this apparent anomaly by investigating the cell-cycle dynamics of DRAD21, the Drosophila orthologue of the Xenopus XRAD21 and Saccharomyces cerevisiae Scc1p/Mcd1p cohesins [3]. Analysis of DRAD21 in S2 Drosophila tissue culture cells and live embryos expressing a DRAD21–green fluorescent protein (GFP) fusion revealed the presence of four distinct subcellular pools of DRAD21: a cytoplasmic pool; a chromosome-associated pool which dissociates from chromatin as chromosomes condense in prophase; a short-lived centrosome-associated pool present during metaphase–anaphase; and a centromere-proximal pool which remains bound to condensed chromosomes, is found along the junction of sister chromatids between kinetochores, and persists until the metaphase–anaphase transition. We conclude that in Drosophila, and possibly all metazoans, a minor pool of cohesin remains bound to centromere-proximal chromatin after prophase and maintains sister-chromatid cohesion until the metaphase–anaphase transition

Estimating genomic instability mediated by Alu retroelements in breast cancer

Alu-PCR is a relatively simple technique that can be used to investigate genomic instability in cancer. This technique allows identification of the loss, gain or amplification of gene sequences based on the analysis of segments between two Alu elements coupled with quantitative and qualitative analyses of the profiles obtained from tumor samples, surgical margins and blood. In this work, we used Alu-PCR to identify gene alterations in ten patients with invasive ductal breast cancer. Several deletions and insertions were identified, indicating genomic instability in the tumor and adjacent normal tissue. Although not associated with specific genes, the alterations, which involved chromosomal bands 1p36.23, 1q41, 11q14.3, 13q14.2, occurred in areas of well-known genomic instability in breast and other types of cancer. These results indicate the potential usefulness of Alu-PCR in identifying altered gene sequences in breast cancer. However, caution is required in its application since the Alu primer can produce non-specific amplification

En quoi l’appartenance à un collectif résultant de l’engagement dans un dispositif externe d’insertion participe-t-elle à la socialisation professionnelle de l’enseignant novice ?

Ce mémoire s’intéresse à l’insertion des enseignants débutants dans la profession et plus précisément à leur socialisation professionnelle et à l’impact que peut avoir un collectif sur ce processus. En effet, les enseignants débutants sont confrontés, dans leurs premières années, à une période de survie (Curran & Goldrick, 2002) dans laquelle ils vivent un choc entre l’image qu’ils ont de la profession et la réalité du terrain (Biémar, 2010). Afin d’aider les novices, la circulaire 5806 (2016), provenant du décret Missions, demande à tous les établissements scolaires de mettre en place, de manière obligatoire à partir de l’année scolaire 2016-2017, un dispositif structurel d’accueil des novices (Circulaire 5806, 2016). Malgré cela, d’autres organismes mettent en place des dispositifs à l’extérieur des établissements scolaires visant à faciliter l’insertion professionnelle des débutants. Cette recherche prend appui sur les travaux de Mukamurera (2012 & 2013), Martineau (2008, 2012 & 2013) ou encore Vallerand (2008) qui se sont questionnés sur les dispositifs d’insertion professionnelle et ont créé des typologies en termes de dispositifs. Il existe au sein des dispositifs externes une dimension collective qui permet le développement de l’identité professionnelle enseignante, ainsi que l’intégration de nouvelles compétences nécessaires à la pratique. Ainsi, la problématique au centre de cette recherche est la suivante : En quoi l’appartenance à un collectif résultant de l’engagement dans un dispositif externe d’insertion participe-t-elle à la socialisation professionnelle de l’enseignant novice ? Afin d’y répondre, une démarche qualitative a été entreprise auprès de trois dispositifs externes : Deux dans la Province de Liège et un dans la Province du Hainaut. Sur base d’entretiens avec les concepteurs, de focus groupes avec les participants et de questionnaires individuels, les données recueillies ont permis de confirmer que les dispositifs permettaient de nombreux apports de différents types chez les débutants et que le collectif semble jouer un rôle prépondérant dans le processus de socialisation professionnelle. En effet, les différents apports sont majoritairement permis par l’entremise du groupe et du climat bienveillant instauré au sein des dispositifs qui permettent aux novices de s’exprimer librement sans crainte d’être jugés. En guise de perspective, il sera intéressant de voir dans quelques années l’impact que pourrait avoir le Pacte d’excellence sur la socialisation professionnelle des débutants grâce aux modifications qui seront apportées à la formation initiale et à l’accompagnement des enseignants débutants dans les écoles.Master [120] en sciences de l'éducation, Université catholique de Louvain, 201

A study of allelic imbalance in human breast cancer.

The analysis of loss of heterozygosity (LOH) in tumours can be used to map candidate sites of tumour-suppressor genes in the human genome. A panel of breast cancer patients was assembled as pairs of tumour and lymphocyte DNA samples and LOH studies carried out by Southern hybridization with polymorphic loci mapping to chromosomes 1 and X. Loci on other chromosomes were also studied as controls. Sequences on chromosome 1 were found to be subject to a variety of alterations creating very complex patterns of rearrangements, including LOH. Deletion mapping identified five independent regions of loss, three of which may correspond to regions identified in previous reports, although individually none occurred with high frequency to support the targeting of a specific locus. The short arm of chromosome 1 primarily underwent interstitial deletions whereas the long arm was subject to both whole arm events (gains and losses) and more localized events. Small deletions on chromosome 1 were found exclusively in patients with poorly-differentiated tumours (i.e. tumours of high grade, p<0.05). A similar study of the X chromosome revealed a high frequency of small regionalized deletions, primarily defining three small independent regions, one located in the distal portion of the pseudoautosomal region of Xp, another spanning the pseudoautosomal boundary into the sex-specific sequences of Xp and a possible third on distal Xq. No gains in copy number were identified at any locus on the X chromosome, in distinct contrast to chromosome 1. Small-scale deletions occurring on the X chromosome were found in patients with no spread of cancer to the lymph nodes, thus correlating with a favourable prognosis (p<0.005). Fluorescence in situ hybridization to formalin-fixed paraffin-embedded tumour sections defined the actual copy number of chromosome 1 for nine tumours. The results obtained confirmed the changes established by Southern hybridization

Loss of Cell Cycle Checkpoint Control in Drosophila Rfc4 Mutants

Two alleles of the Drosophila melanogaster Rfc4(DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these twoDmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes

A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity

Coordination between separate pathways may be facilitated by the requirements for common protein factors, a finding congruent with the link between proteins regulating DNA replication with other important cellular processes. We report that the smallest of Drosophila origin recognition complex subunits, Orc6, was found in embryos and cell culture localized to the cell membrane and cleavage furrow during cell division as well as in the nucleus. A two-hybrid screen revealed that Orc6 interacts with the Drosophila peanut (pnut), a member of the septin family of proteins important for cell division. This interaction, mediated by a distinct C-terminal domain of Orc6, was substantiated in Drosophila cells by coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6 expression with double-stranded RNA resulted in a formation of multinucleated cells and also reduced DNA replication. Deletion of the C-terminal Orc6–peanut interaction domain and subsequent overexpression of the Orc6 mutant protein resulted in the formation of multinucleated cells that had replicated DNA. This mutant protein does not localize to the membrane or cleavage furrows. These results suggest that Orc6 has evolved a domain critical mainly for cytokinesis

Functional analysis of mutant and wild-type Drosophila origin recognition complex

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes