Persistence of DNA threads in human anaphase cells suggests late completion of sister chromatid decatenation

PICH (Plk1-interacting checkpoint helicase) was recently identified as an essential component of the spindle assembly checkpoint and shown to localize to kinetochores, inner centromeres, and thin threads connecting separating chromosomes even during anaphase. In this paper, we have used immuno-fiber fluorescence in situ hybridization and chromatin-immunoprecipitation to demonstrate that PICH associates with centromeric chromatin during anaphase. Furthermore, by careful analysis of PICH-positive anaphase threads through FISH as well as bromo-deoxyurdine and CREST labeling, we strengthen the evidence that these threads comprise mainly alphoid centromere deoxyribonucleic acid. Finally, by timing the addition of ICRF-193 (a specific inhibitor of topoisomerase-II alpha) to cells synchronized in anaphase, we demonstrate that topoisomerase activity is required specifically to resolve PICH-positive threads during anaphase (as opposed to being required to prevent the formation of such threads during earlier cell cycle stages). These data indicate that PICH associates with centromeres during anaphase and that most PICH-positive threads evolve from inner centromeres as these stretch in response to tension. Moreover, they show that topoisomerase activity is required during anaphase for the resolution of PICH-positive threads, implying that the complete separation of sister chromatids occurs later than previously assumed

Models for congenital generalized lipodystrophy, type 1 and type 2 (CGL1, CGL2) in Drosophila : functional analysis of BSCL2 and AGPAT2

Die kongenitale generalisierte Lipodystrophie (CGL) ist eine seltene autosomal-rezessiv vererbte Erkrankung, die durch die vollständige Abwesenheit von Fettgewebe von Geburt oder frühen Kindheit an, einer schweren Insulinresistenz und einem frühen Auftreten von Diabetes gekennzeichnet ist. Von den 4 Genen, von denen derzeit bekannt ist, dass sie CGL verursachen können, ist der Krankheitsmechanismus hinter der AGPAT2-Mutation (CGL1) bekannt, während Mutationen in BSCL2 zwei stark unterschiedliche Phänotypen verursachen können: autosomal-rezessive CGL2 und autosomal dominant hereditäre Neuropathie (SPG17). Obwohl in den letzten Jahren viel Forschung an BSCL2 betrieben wurde, bleiben die molekulare Funktionen, die zu den jeweiligen Phänotypen führt, ungewiss.Zur Entwicklung von Krankheitsmodellen erwies sich der Modellorganismus Drosophila melanogaster als nützlich, da die homologen Gene CG9904 (dBSCL2) und CG3812 (dAGPAT2) stark konserviert sind. Die Mutationsstellen für den neurologischen Phänotyp SPG17 sind jedoch nicht in dBSCL2 konserviert, was die Nützlichkeit zur Untersuchung einschränkt. Unter Verwendung von Exzisionsmutagenese konnten mehrere CG9904-Nullmutantenstämme etabliert werden. Leider erwies sich die CG3812-Mutagenese als nicht erfolgreich. Zusätzlich wurden transgene Stämme, die dBSCL2-EGFP-Fusionsproteine enthielten, erzeugt. Dies bildete die Grundlage für die phänotypische Charakterisierung und die Untersuchung der subzellulären Verteilung von dBSCL2.Da die BSCL2-Nullmutation beim Menschen zu einem schweren Phänotyp des Fettgewebes (CGL2) führt, lag der Schwerpunkt der phänotypischen Charakterisierung des dBSCL2-Knock-outs auf Stoffwechsel-Assays. Der beobachtete Phänotyp war durch eine verminderte Resistenz gegen Hunger, eine Verschiebung des Lipidgehaltes und eine veränderte Morphologie der Lipid-Tröpfchen gekennzeichnet. Dies zeigt, dass das Drosophila dBSCL2-Modell geeignet ist, die Pathogenese des menschlichen Phänotyps aufzuklären.Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder characterized by near complete absence of adipose tissue from birth or early infancy, severe insulin resistance, and early onset of diabetes. Among the four genes currently known to cause CGL, the disease mechanisms behind AGPAT2 mutation (CGL1, encodes 1-acylglycerol-3-phosphate O-acyltransferase-2) are known, whereas mutations in BSCL2 can cause two distinct phenotypes: autosomal recessive congenital lipodystrophy type 2 (CGL2) and autosomal dominant hereditary neuropathy (SPG17). Although much work has been done on BSCL2 in the recent years, the detailed molecular function resulting in these phenotypes remains uncertain. To develop disease models, the model organism Drosophila melanogaster proved useful, as the homologous genes CG9904 (dBSCL2) and CG3812 (dAGPAT2) encode proteins highly similar to the human proteins. However, the mutation sites for SPG17 are not conserved in dBSCL2, limiting the usefulness for studying the neurologic phenotype. Using excision mutagenesis, multiple CG9904 null mutant strains could be established. Unfortunately, the CG3812 screen proved to be unsuccessful in generating null mutant strains. In addition, transgenic strains containing dBSCL2-EGFP-fusions proteins were generated. This constituted the basis for the phenotypic characterisation and to study the tissue and sub-cellular distribution of dBSCL2. Because the BSCL2 null mutation in human results in a severe phenotype of the adipose tissue (CGL2), the focus of the phenotypic characterization of the dBSCL2 knock-out was on metabolic assays like hatching rate, starvation and lipid droplet formation. The observed phenotype was marked by reduced resistance to starvation, a shift in lipid content and altered lipid droplet morphology. This shows that the Drosophila dBSCL2-model is suitable to study the proteins function and to elucidate the pathogenesis of the human phenotype.eingereicht von Thomas SchwarzbraunZusammenfassungen in Deutsch und EnglischKarl-Franzens-Universität Graz, Dissertation, 2017OeBB(VLID)230493

A 19-Mb <i>de novo</i> deletion on BTA 22 including <i>MITF </i>leads to microphthalmia and the absence of pigmentation in a Holstein calf

Mutations in MITF lead to a large variety of phenotypes in human, mice and other species. They mostly affect pigmentation and hearing, whereas in mice, they may additionally cause microphthalmia and osteopetrosis. In this study, we report a single case of a Holstein calf with lack of pigmentation and microphthalmia born to healthy parents. Mendelian analysis of high-density SNP genotypes reveals a large number of parentage errors showing missing paternal alleles in the offspring, indicating a deletion encompassing 19 Mb on BTA 22. The genomic deletion affects the paternal allele and includes MITF and 131 other annotated genes. As the calf shows only one copy of the BTA 22 segment, the observed phenotype is probably caused by haploinsufficiency of the genes in that genomic region. Both the observed lack of skin pigmentation and reduced eye size can most likely be explained by a lack of MITF function

Questioni di diritto sostanziale e tributario connesse al riconoscimento del trust nell'ordinamento italiano. Parte seconda

in "Fiducia e trust" n. 2/2002, allegato a "Il fisco" n. 34/200