No abstract

During meiosis, sister chromatid cohesion is required for normal levels of homologous recombination, although how cohesion regulates exchange is not understood. Null mutations in orientation disruptor (ord) ablate arm and centromeric cohesion during Drosophila meiosis and severely reduce homologous crossovers in mutant oocytes. We show that ORD protein localizes along oocyte chromosomes during the stages in which recombination occurs. Although synaptonemal complex (SC) components initially associate with synapsed homologues in ord mutants, their localization is severely disrupted during pachytene progression, and normal tripartite SC is not visible by electron microscopy. In ord germaria, meiotic double strand breaks appear and disappear with frequency and timing indistinguishable from wild type. However, Ring chromosome recovery is dramatically reduced in ord oocytes compared with wild type, which is consistent with the model that defects in meiotic cohesion remove the constraints that normally limit recombination between sisters. We conclude that ORD activity suppresses sister chromatid exchange and stimulates inter-homologue crossovers, thereby promoting homologue bias during meiotic recombination in Drosophila.

Angelman syndrome (AS) is caused by chromosome 15q11-q13 deletions of maternal origin, by paternal uniparental disomy (UPD) 15, by imprinting defects, and by mutations in the UBE3A gene. UBE3A encodes a ubiquitin-protein ligase and shows brain-specific imprinting. Here we describe UBE3A coding-region mutations detected by SSCP analysis in 13 AS individuals or families. Two identical de novo 5-bp duplications in exon 16 were found. Among the other 11 unique mutations, 8 were small deletions or insertions predicted to cause frameshifts, 1 was a mutation to a stop codon, 1 was a missense mutation, and 1 was predicted to cause insertion of an isoleucine in the hect domain of the UBE3A protein, which functions in E2 binding and ubiquitin transfer. Eight of the cases were familial, and five were sporadic. In two familial cases and one sporadic case, mosaicism for UBE3A mutations was detected: in the mother of three AS sons, in the maternal grandfather of two AS first cousins, and in the mother of an AS daughter. The frequencies with which we detected mutations were 5 (14%) of 35 in sporadic cases and 8 (80%) of 10 in familial cases.

Drosophila Nipped-B is an essential protein that has multiple functions. It facilitates expression of homeobox genes and is also required for sister chromatid cohesion. Nipped-B is conserved from yeast to man, and its orthologs also play roles in deoxyribonucleic acid repair and meiosis. Mutation of the human ortholog, Nipped-B-Like (NIPBL), causes Cornelia de Lange syndrome (CdLS), associated with multiple developmental defects. The Nipped-B protein family is required for the cohesin complex that mediates sister chromatid cohesion to bind to chromosomes. A key question, therefore, is whether the Nipped-B family regulates gene expression, meiosis, and development by controlling cohesin. To gain insights into Nipped-B's functions, we compared the effects of several Nipped-B mutations on gene expression, sister chromatid cohesion, and meiosis. We also examined association of Nipped-B and cohesin with somatic and meiotic chromosomes by immunostaining. Missense Nipped-B alleles affecting the same HEAT repeat motifs as CdLS-causing NIPBL mutations have intermediate effects on both gene expression and mitotic chromatid cohesion, linking these two functions and the role of NIPBL in human development. Nipped-B colocalizes extensively with cohesin on chromosomes in both somatic and meiotic cells and is present in soluble complexes

NorDxWith the commercial availability of new high-risk HPV screening assays, we sought to compare a real-time PCRbased assay with our current signal amplification assay. The Cobas 4800 HPV assay (Roche Molecular Diagnostics, Indianapolis, IN) recently received FDA approval for detection of 14 HR-HPV genotypes: HPV-16 and HPV-18 are each identified separately and the other 12 HR-HPV genotypes are identified in a pool. The Hologic Cervista HR-HPV reagents (Hologic, Bedford, MA) currently used by our laboratory detect 14 HR-HPV genotypes in 3 different groups or clades (A5/A6, A7, and A9). Samples positive for the HR-HPV screening assay are reflexed to the Hologic Cervista 16/18 genotype assay if requested by the clinician. The purpose of this study is to compare the Roche assay with our current method. We selected 272 gynecologic specimens collected in PreservCyt (Hologic) for this study. Cytology was performed, followed by evaluation of HR-HPV infection. Agreement between the 2 HPV assays was 83%. Of the 46 discrepant samples, the majority (52%) were negative by Roche and positive for all 3 HPV virus clades on the Hologic platform (>3.0 HPV-fold over zero, minimum fold over zero value for HPV positivity, 1.93). These "triple-positive" specimens all had a very high DNA content with an average DNA fold over zero between 18 and 25; the minimum valid value is 1.5-fold over zero for this assay. When the same specimens were diluted in PreservCyt and tested again on the Hologic platform, valid negative results were obtained. Some of these triple-positive samples were sent to a reference laboratory for confirmatory testing by a third method (Qiagen, Valencia, CA), and, again, all were negative. These results indicate that the triple-positive results obtained are falsepositives, confirming previous clinical observations showing high discrepancy rates between cytology and Hologic HPV results.