The maintenance of eusociality in neotropical, swarm-founding wasps is difficult to explain because brood relatedness is predicted to be very low due to the presence of multiple queens in each colony and because workers are not morphologically constrained from laying eggs. Analysis of 4 morphological measures revealed no physical reasons why workers could not lay eggs in Polybia emaciata. A large variance in queen number, high queen relatedness, and a significant negative correlation between mean queen number and number of eggs per queen strongly suggest that cyclical oligogyny is probably the most important, and possibly the only, mechanism maintaining high relatedness in P. emaciata. Preferential care by workers of closely related kin on a discrete section of a comb is another possible mechanism that requires further research. However, inbreeding and unequal egg-laying by active queens are not important in elevating relatedness in P. emaciata.

Compensation is a mechanism by which Drosophila melanogaster can increase its ribosomal RNA repeat unit multiplicity by up to two-fold. The phenomenon occurs under defined genetic conditions, is restricted to the X chromosome nucleolus organizer region, and occurs only in somatic cells of both sexes; therefore the increase is not transmitted to progeny. Molecular analysis was used to analyze which types of repeat units were disproportionately replicated during compensation. It was found that the relative percentages of uninterrupted repeats and those containing major or minor type I insertions were the same as for the normal X chromosome. This nonselective amplification of ribosomal RNA repeat units distinguishes compensation as a mechanism distinct from independent ribosomal DNA polytenization. Five Y chromosome nucleolus organizer mutants generated by ethyl methanesulfonate mutagenesis of the B('s)Ybb('+)y('+) chromosome were previously analyzed as to ribosomal RNA repeat unit content, hemizygous phenotype and behavior in additivity tests with rDNA-deficient X chromosomes. Molecular analysis of the repeat unit organization of four of these mutants showed a direct correlation between their uninterrupted repeat unit number, phenotype, and functionality, as evidenced by the additivity tests. These constitute a mutant class in which greater numbers of uninterrupted repeats resulted in decreased severity of bobbed phenotypes. Another mutant allele, bb('1-4), failed to show additivity and was an early developmental lethal despite an uninterrupted repeat multiplicity sufficient to reach the late pupal stage. This apparent suppression of presumably functional repeats is indicative of a separate mutant class for the Y chromosome nucleolus organizer redundant locus.

Bacillus subtilis bacteriophage SP01 causes pleiotrophic cessations of host metabolism and kills the host. Nonetheless, a large number of restriction fragments, representing approximately two thirds of the genome, can be maintained as cloned fragments in the natural host. Several fragments, which were not easily cloned, contain genes that are detrimental to bacterial hosts. The clones are convenient sources of specific SP01 fragments for use in restriction mapping and were used to map the Xba I sites in the SP01 genome. Specific genes can be identified on cloned fragments by interactions between phage mutants and cloned fragments during infection. Most of the known genes were located on specific fragments, and this information was used to refine the correlation between the physical and genetic maps of SP01. SP01 DNA contains a thymine analog, hydroxymethyl uracil. Cloned fragments contain thymine, even during SP01 infection, yet cloned DNA is active in expression of its genetic information and in recombination with superinfecting SP01 DNA. These results argue that hydroxymethyl uracil is not strictly required for those activities.

Vesico-ureteric reflux (VUR) is a congenital urinary tract defect that results in the retrograde flow of urine from the bladder to the kidneys. It is a defect in the formation of the uretero-vesical junction such that the ureter does not insert properly into the bladder. VUR affects up to 1% of the population and patients are at increased risk of developing recurrent urinary tract infections, hypertension, and end-stage renal disease. Patients with VUR frequently have malformed kidneys and short intravesical ureters, one component of the uretero-vesical junction. The co-occurrence of these defects suggests that they arise from a common developmental mechanism, notably, abnormal formation of the ureteric bud. The ureteric bud develops into both the kidney and the urinary tract. We hypothesize that proper ureteric bud development is critical for the formation of a normal kidney and an intact uretero-vesical junction. We identified VUR in two mouse lines at birth: the Pax21Neu+/- mouse and the inbred C3H/HeJ mouse. At postnatal day 1, Pax21Neu+/- and C3H/HeJ mice had a 32% and 100% incidence of VUR, respectively. Control mice (CD1 and C57BL/6J) had a 6.25% and 0% incidence of VUR, respectively. We measured kidney size in all mice tested for VUR and Pax21Neu+/- mice had small malformed kidneys while C3H/HeJ mice had small but no malformed kidneys at birth when compared to controls. Both refluxing mouse models had significantly shorter intravesical ureters compared to controls, providing evidence for a defective uretero-vesical junction. To understand the embryonic origin of VUR, we characterized the position of the ureteric bud along the mesonephric duct. At embryonic day 10.5, both Pax21Neu+/- and C3H/HeJ embryos had ureteric buds that exited from a more caudal position along the mesonephric duct compared to controls. Furthermore, detailed analysis of the developing kidney and urinary tracts revealed that b / Le reflux vésico-rénal est un défaut de l'appareil urinaire, dontl'urine remonte anormalement depuis la vessie vers les reins. Le reflux est causé par un défaut dans la formation de la jonction entre la vessie et l'uretère. Le reflux vésico-rénal est une anomalie génétique humaine, affectant près de 1% de la population. Les patients sont susceptibles de développer des infections urinaires, de l'hypertension, et de la néphropathie. Ils sont aussi sujets à des malformations rénales et peuvent présenter des anomalies au niveau de la jonction entre la vessie et l'uretère (uretères intravésicaux courts). Étant donné que ces malformations se manifestent ensemble, il a été suggéré qu'elles résultent toutes d'un mécanisme commun. Les reins et l'appareil urinaire se développent à partir du bourgeon urétéral. Nous présumons que le développement normal du bourgeon urétéral est important pour la formation adéquate aussi bien du rein que de la jonction entre la vessie et l'uretère. Deux lignées de souris atteintes de reflux vésico-rénal ont été identifiées: la souris Pax21Neu+/- et la souris consanguine C3H/HeJ. À la naissance, les souris Pax21Neu+/- et C3H/HeJ avaient une incidence de reflux vésico-rénal, respectivement, de 32% et 100%. Les souris qui servaient de contrôle (CD1 et C57BL/6J) avaient, respectivement, une incidence de 6.25% et 0%. Lorsque nous avons mesuré la taille des reins pour toutes les souris, nous nous sommes aperçus que les souris Pax21Neu+/- et C3H/HeJ avaient des reins plus petits comparativement aux contrôles. Elles avaient aussi des uretères intravésicaux plus courts que ceux des contrôles, ce qui confirme la présence d'un défaut dans la formation de la jonction entre la vessie et l'uretère. Nous avons également caractérisé la position du bourgeon urétéral. Au stade embryonnaire 10.5, les bourgeons urétéraux des souris Pax21Neu+

Genomic imprinting is a phenomenon that results in parent of origin dependant gene expression. During mammalian gametogenesis, genomic imprints are erased, and reestablished in sperm and oocytes in a sex-dependent manner. Most genomic imprints that are inherited by the embryo from its parents are maintained throughout the life of the individual. Aberrant genomic imprinting may lead to embryonic lethality, developmental anomalies and increased risk of cancer. To better understand the mechanisms of imprinting errors, we explored two potential sources of such errors: a) genetic anomalies that affect DNA methylation and spermatogenesis in mice, and b) the influence of cell culture on imprinting maintenance in human somatic cells. We found that heterozygous mutations in murine Mthfr, Dnmt1, Dnmt3a/3b, Spo11, and Msh5 that are known to affect DNA methylation or meiotic recombination have no effect on imprint establishment of H19 and the intragenic differentially methylated region (IGDMR) in mature sperm. Next, we tested the hypothesis that local chromosomal anomalies such as deletions or translocations may impair imprint establishment using two mouse models, mice with an H19 DMR deletion and carriers of Robertsonian translocations. We found incomplete establishment of H19 DMR methylation in the sperm of heterozygous carriers of a Robertsonian translocation, Rb 8.12, that suggests a mechanistic link between chromosomal anomalies and imprint establishment or maintenance errors. DNA methylation patterns are often poorly conserved through cell culture. This could significantly impact gene expression profiles. To investigate the effect of cell culture on DNA methylation profiles, we analyzed methylation in the differentially methylated domains of 5 imprinted regions: the IGDMR; potassium voltage-gated channel, KQT-like subfamily, member 1, (KCNQ1); small nuclear ribonucleoprotein polypeptide N (SNRPN), mesoderm spe / RÉSUMÉL'empreinte génomique est un processus biologique où l'expression d'un gène dépend de son origine parentale. Chez les mammifères, au cours de la gamétogenèse, les empreintes génomiques sont supprimées puis rétablies dans les spermatozoïdes et les ovules, en fonction du sexe. La plupart des empreintes génomiques qui sont héritées des parents sont maintenues tout au long de la vie de chaque individu. Une empreinte génomique aberrante peut induire une létalité embryonnaire, des développements anormaux ou augmenter le risque d'apparition de cancer. Afin de mieux comprendre comment se produisent ces erreurs, nous avons étudié deux mécanismes potentiellement impliqués dans l'apparition d'empreintes erronées: a) les anomalies génétiques qui perturbent la méthylation de l'ADN ou la spermatogenèse chez la souris, et b) l'influence de la culture cellulaire sur le maintien de l'empreinte dans les cellules somatiques humaines. Nous avons déterminé que, chez la souris, les mutations à l'état hétérozygote de Mthfr, Dnmt1, Dnmt3a/3b, Spo11 et Msh5, qui sont connues pour altérer la méthylation de l'ADN ou la recombinaison méiotique, n'ont aucun effet sur l'établissement de l'empreinte au niveau du gène H19 et de la région intragenic differentially methylated region (IGDMR) dans le sperme. Nous avons ensuite cherché à déterminer si des anomalies chromosomiques locales comme les délétions ou les translocations pouvaient perturber l'établissement de l'empreinte. Pour tester cette hypothèse, nous avons utilisé deux modèles murins : une lignée de souris portant une délétion au niveau de la région différentiellement méthylées H19 DMR et une lignée de souris portant des translocations robertsoniennes. Nous avons mis en évidence que le sperme de souris hétérozygotes pour la translocation robertsonienne Rb 8.12 présente un défaut dans l'établissement de la mé

The claudin family of proteins is required for the formation of tight junctions between epithelial cells. Tight junctions form uninterrupted paracellular barriers on the apical surface linking adjacent epithelial cells. As a result, they promote cell-cell adhesion and regulate the paracellular flow of soluble ions. During kidney development, an epithelial outgrowth of the nephric duct called the ureteric bud (UB) emerges and invades the neighboring metanephric mesenchyme where it undergoes a series of branching events in a process known as branching morphogenesis. It has been shown that claudin-3 (Cldn3) and claudin-7 (Cldn7) transcripts are upregulated in the ureteric bud (UB) versus the metanephric mesenchyme (MM) during kidney development. We hypothesize that if Cldn3 and Cldn7 form tight junctions in the epithelial UB, they will determine the pattern of UB branching. Using transmission electron microscopy, we have established that tight junctions are situated between epithelial cells of the UB that undergo branching. Whole-mount in situ hybridization assays established that Cldn3 and Cldn7 transcripts are expressed in the UB at embryonic day (E)10.5, 13.5 and 16.5. Double immunofluorescence experiments revealed that CLDN3 is localized to tight junctions at the apical domain of UB cells, while CLDN7 is predominately expressed on the basolateral membrane. To determine the functional role of these claudins, we took advantage of the mIMCD-3 cell culture model of tubulogenesis. The mIMCD-3 cell line is derived from the embryonic UB, and when placed in a type-I collagen matrix these cells undergo tubulogenesis and branching in a manner morphologically similar to the UB. Double immunofluorescence and Z-stacking showed that mIMCD-3 cells express both CLDN3 and CLDN7 at the tight junction. Stable cell lines expressing either CLDN3 or CLDN7 fused at the N-terminus to the red fluorescent protein (RFP) mCherry w / La famille de protéines claudine est nécessaire pour la formation des jonctions serrées entre les cellules épithéliales. Les jonctions serrées forment des obstacles paracellulaires ininterrompus sur la surface apicale entre les cellules épithéliales adjacentes. En conséquence, ils favorisent l'adhérence cellule-cellule et régularisent le transport des ions solubles paracellulaire. Au cours du développement du rein, une excroissance épithéliale du canal nephric appelée urétérale bourgeon (UB) se dégage et envahit le mésenchyme métanephrique (MM) voisins où il subit une série de manifestations de branchement dans un processus connu sous le nom de la morphogenèse de ramification. Il a été démontré que claudin-3 et claudin-7 transcriptions sont augmentées dans l'UB par rapport au MM au cours du développement du rein. Nous faisons l'hypothèse que, si Cldn3 et Cldn7 forment les jonctions serrées dans l'épithéliales UB, ils détermineront le type de ramification UB.En utilisant la microscopie électronique en transmission, nous avons établi que des jonctions serrées sont situés entre les cellules épithéliales de l'UB qui subissent ramification. L'hybridation in situ établi que claudin-3 et claudin-7 sont exprimés en UB à jour embryonnaire (E) 10,5, 13,5 et 16,5. Double immunofluorescence a révélée que la protéine Cldn3 est localisée à des jonctions serrées au domaine apical de l'UB, tandis que Cldn7 est surtout exprimé sur la membrane basolatérale. Pour déterminer le rôle fonctionnel de ces claudins, nous avons profité de la mIMCD-3 modèle de la culture cellulaire de la formation de tubules. Le mIMCD-3 lignée cellulaire est issue de l'embryon de UB, et lorsqu'il est placé dans un type-I matrice collagène, ces cellules commence à former des tubules et une ramification d'une manière morphologiquement semblables à l'UB. Double immunofluorescence et des

Introduction Leprosy is a disease caused by Mycobacterium leprae that affects an estimated 300,000 new cases each year. The LTA+80 A promoter allele, a strong leprosy risk factor (Alcais et al. 2007), has been shown to facilitate binding of the ABF1 transcriptional repressor in human B-cells (Knight et al. 2004). Direct studies on the impact of LTA+80 genotypes on LTA expression levels following exposure of human cells to M. leprae are missing. Results Comparative analysis of LTA expression in M. leprae stimulated and un-stimulated human blood samples revealed that LTA expression patterns did not differ between genotype groups. Observed LTA expression differences show a variable pattern within genotype groups suggesting a complex mechanism of action. Using a transient transfection assay, the LTA+80A allele was shown to exert significantly reduced activity relative to the "C" allele (P=0.01) in a Ramos B-lymphocyte cell line. These results follow the hypothesis that the LTA+80A allele has a repressing affect on expression. However, when the alleles were co-transfected with ABF1, there was no alteration of baseline expression levels, suggesting that ABF1 is not responsible for differential ability of the LTA promoter alleles to drive luciferase activity. Analysis of allele-specific expression in heterozygous cell lines using cross-linking experiments indicated that expression was not LTA+80 allele-specific as the allelic ratios were equal in presence and absence of cross-linking reagent. In addition, quantitative RT-PCR analysis of the two reporter constructs did not reveal an expression difference at the level of RNA alleles. Studies on posttranscriptional regulation indicated that LTA+80 does not affect translational efficiency, but rather impacts on regulation at the level of nuclear export, where LTA+80C is preferentially found in the cytoplasm (P=0.01). Further analysis, suggests LTA uses the CRM1 exp / IntroductionLa lèpre est une maladie causée par Mycobacterium leprae qui engendre environ 300 000 nouveaux cas chaque année. Il a été démontré que l'allèle A du promoteur de LTA+80, un important facteur de risque pour la lèpre (Alcais et al. 2007) se lie de manière sélective au répresseur de transcription ABF1 dans les cellules B humaines (Knight et al. 2004). Des études directes de l'impact des génotypes de LTA+80 sur l'expression de LTA suite à l'exposition de cellules humaines à l'antigène de M.leprae sont manquantes.RésultatsL'analyse comparative de l'expression de LTA dans les échantillons sanguins stimulés ou non-stimulés révèlent que l'expression des niveaux de LTA ne change pas d'un groupe génotypique à l'autre. Les differences d'expression de LTA observées se présentent sous un schéma variable à l'intérieur des groupes génotypiques ce qui indique un mécanisme d'action complexe. À l'aide d'un test de transfection transitoire, il fut montré que l'allèle « A » de LTA+80 stimulait signifivativement moins l'activité du rapporteur luciférase comparativement à l'allèle « C » (P=0.01) chez des lymphocytes B Ramos. Ces résultats vont de pair avec l'hypothèse selon laquelle l'expression de l'allèle A de LTA+80 est réprimée sélectivement. Cependant, lorsque les deux allèles de LTA+80 furent co-transfectées avec ABF1, aucune variation des niveaux d'expression de base ne fut observée, ce qui suggère que ABF1 n'est peut-être pas responsable de la capacité différentielle des allèles du promoteur de LTA à faire varier l'activité luciférase. Une analyse de l'expression spécifique à chaque allèle dans des lignées cellulaires hétérozygotes à l'aide d'expériences de pontage de la chromatine a indiqué que l'expression n'est pas spécifique aux allèles de LTA+80. Une analyse par RT-PCR quantitatif des deux constructions n'a pas révélé de différenc

We have isolated, cloned, and sequenced, from the genome of human (HeLa) cells, a 1.797 Kb EcoR1 satellite II DNA fragment that displayed partial identity to the Drosophila melanogaster transposable P-element. / The sequence analysis of this clone (pKS36) indicated that it has originated from the tandem amplification of pentameric repeats that were derived from satellite II and III canonical consensus sequence 5$ sp prime$ TTCCA 3$ sp prime$. A two-stage decay mechanism, based on the methylation and subsequent deamination of cytosine residues within the pentamers, was proposed to explain the non-random base substitutions that were observed in pKS36, as well as in other sequenced members of the satellite II and III DNA families. In addition, this two-stage decay model correlated with the Tag1 and Hinf1 polymorphisms that were observed between related satellite DNA members. / Clone pKS36 also contains a region of 49 bp devoid of satellite sequences that was found, by southern hybridization, to be present in pKS36 closely related (pKS36-like) but absent from more divergent (pKS36-related) cloned satellite II and III DNAs. pKS36-related satellite DNAs represent up to 2% of the genomes of HeLa and MeWo cells, and are organized mostly in tandem arrays of 1.8 Kb EcoR1, Kpn1, and Sau3A DNA fragments. pKS36-like satellite DNAs represent less than 1% of the genome of HeLa cells, and are found mainly organized as 1.65 Kb, 1.95 Kb and 3.6 Kb EcoR1 elements, though their Kpn1 and Sau3A distributions resemble that of pKS36-related satellite DNAs. Cell specific organization of satellite DNAs, that may be the result of chromosomal translocations inherent to cultured cells, was observed in the two human (HeLa and MeWo) cell lines. / The analysis, by southern hybridization, of satellite DNAs using field inversion gel electrophoresis revealed the presence, in HeLa cells, of satellite DNA clusters ranging from 150 Kb to 500 Kb in length. / Using rodent-human hybrid cell DNAs, the members of the pKS36 satellite II DNA family were found to reside mainly on human chromosomes 7, 12, 14, 15, 16, and 22.

The human androgen receptor (hAR) is a member of the steroid receptor family of nuclear receptors (NRs) that acts as a ligand-activated transcription factor. Mutations in the androgen receptor impair normal male sexual development resulting in a clinical continuum of virilization disorders known as the androgen insensitivity syndrome (AIS). Characterization of key AR mutations has enabled a better understanding of structure-function relationships of the AR, and serves as a prototype for steroid receptor mechanisms of action. / NH2-/COOH-terminal (N/C-terminal) interactions appear to be critical for AR function in vivo as evidenced by the association of AIS with single amino acid mutations that disrupt the N/C-interaction. / In the first paper, we examine the effects of five distinct C-terminal mutations on hAR N/C-interactions in the presence and absence of the hAR coactivator, TIF2. We found that each of the mutations disrupted normal hAR N/C-interactions, partially explaining the molecular defects underlying the corresponding AIS phenotypes. Interestingly, we show a strong correlation between the strength of N/C-interaction and the phenotypic degree of AIS observed. / In the second paper, we investigated the role of N-terminal mutations on hAR N/C-interactions, in the presence and absence of TIF2. While there have been no previous reports of the effects of N-terminal hAR mutations on N/C-interaction, we show that two distinct mutations in this terminal of the hAR are directly responsible for the disruption of normal N/C-interaction in a patient with complete androgen insensitivity. We suggest that these mutations affect the N/C-coactivator-binding interface, thus reducing the overall transcriptional competence of the hAR.

DNA methylation constitutes an epigenetic modification of DNA that is initiated during gametogenesis and is essential for normal development. Methylation is postulated to be the molecular mark underlying genomic imprinting whereby genes are differentially expressed according to parental origin. The loss of the oocyte-derived DNMT1o maintenance methyltransferase affects the methylation imprints during preimplantation development, producing epigenetic mosaic embryos. Here we show that embryos that develop in the absence of DNMT1o demonstrate profound phenotypic variation in both fetal and extraembryonic tissues, as well as severe pregnancy loss by midgestation. Morphological assessment was complemented with methylation analysis of fetal and extraembryonic tissues at the differentially methylated domain (DMD) of the imprinted gene Snrpn. The general conclusion from this study is that extragenetic programming in the embryo greatly influences pregnancy outcome and phenotype in mouse embryos. The relevance of this work to human imprinting disorders and cloning technologies is discussed.