Draft Genome Sequences of Nine Clinical Isolates of Vancomycin-Resistant Enterococci.

In 2012, there was an increase in vancomycin-resistant enterococci (VRE) isolated from the intensive care unit at the University Hospital of Cologne. Using whole-genome sequencing it was possible to establish that bloodstream infections with VRE were not the result of an outbreak or cross infections.

Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos.

Cloned bovine preimplantation embryos were generated by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts with a silent copy of the pluripotency reporter gene GOF, integrated at a single site of a chromosome 13. GOF combines the regulatory Oct4/Pou5f1 sequence with the coding sequence for EGFP. EGFP expression served as a marker for pluripotency gene activation and was consistently detected in preimplantation embryos with 9 and more cells. Three-dimensional radial nuclear positions of GOF, its carrier chromosome territory and non-carrier homolog were measured in nuclei of fibroblasts, and of day 2 and day 4 embryos, carrying 2 to 9 and 15 to 22 cells, respectively. We tested, whether transcriptional activation was correlated with repositioning of GOF toward the nuclear interior either with a corresponding movement of its carrier chromosome territory 13 or via the formation of a giant chromatin loop. A significant shift of GOF away from the nuclear periphery was observed in day 2 embryos together with both carrier and non-carrier chromosome territories. At day 4, GOF, its carrier chromosome territory 13 and the non-carrier homolog had moved back toward the nuclear periphery. Similar movements of both chromosome territories ruled out a specific GOF effect. Pluripotency gene activation was preceded by a transient, radial shift of GOF toward the nuclear interior. The persistent co-localization of GOF with its carrier chromosome territory rules out the formation of a giant chromatin loop during GOF activation.

Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos.

Nuclear landscapes were studied during preimplantation development of bovine embryos, generated either by in vitro fertilization (IVF), or generated as cloned embryos by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts, using 3-dimensional confocal laser scanning microscopy (3D-CLSM) and structured illumination microscopy (3D-SIM). Nuclear landscapes of IVF and SCNT embryonic nuclei were compared with each other and with fibroblast nuclei. We demonstrate that reprogramming of fibroblast nuclei in cloned embryos requires changes of their landscapes similar to nuclei of IVF embryos. On the way toward the 8-cell stage, where major genome activation occurs, a major lacuna, enriched with splicing factors, was formed in the nuclear interior and chromosome territories (CTs) were shifted toward the nuclear periphery. During further development the major lacuna disappeared and CTs were redistributed throughout the nuclear interior forming a contiguous higher order chromatin network. At all stages of development CTs of IVF and SCNT embryonic nuclei were built up from chromatin domain clusters (CDCs) pervaded by interchromatin compartment (IC) channels. Quantitative analyses revealed a highly significant enrichment of RNA polymerase II and H3K4me3, a marker for transcriptionally competent chromatin, at the periphery of CDCs. In contrast, H3K9me3, a marker for silent chromatin, was enriched in the more compacted interior of CDCs. Despite these striking similarities, we also detected major differences between nuclear landscapes of IVF and cloned embryos. Possible implications of these differences for the developmental potential of cloned animals remain to be investigated. We present a model, which integrates generally applicable structural and functional features of the nuclear landscape.

FISH on 3D preserved bovine and murine preimplantation embryos.

Fluorescence in situ hybridization (FISH) is a commonly used technique for the visualization of whole chromosomes or subchromosomal regions, such as chromosome arms, bands, centromeres, or single gene loci. FISH is routinely performed on chromosome spreads, as well as on three-dimensionally preserved cells or tissues (3D FISH). We have developed 3D FISH protocol for mammalian preimplantation embryos to investigate the nuclear organization of chromosome territories and subchromosomal regions during the first developmental stages. In contrast to cells, embryos have much more depth and their nuclei are therefore less accessible to probes used to visualize specific genomic regions by FISH. The present protocol was developed to establish a balance between sufficient embryo permeabilization and maximum preservation of nuclear morphology.

Changes of higher order chromatin arrangements during major genome activation in bovine preimplantation embryos.

Gene-dense chromosome territories (CTs) are typically located more interior, gene-poor CTs more peripheral in mammalian cell nuclei. Here, we show that this gene-density correlated CT positioning holds for the most gene-rich and gene-poor bovine chromosomes 19 and 20, respectively, in bovine fibroblast and lymphocyte nuclei. In order to determine the period at which this non-random CT order is established during development, we performed fluorescence in situ hybridization, on three-dimensionally preserved bovine preimplantation embryos generated by in vitro fertilization and investigated the distribution of BTA 19 and 20 CTs. Radial arrangements of CTs 19 and 20 were the same up to the 8-cell stage. At the 10- to 16-cell stage, however, a significant difference became apparent with CTs 19 localized more internally and CTs 20 more peripherally. Since major genome activation in bovine embryos occurs at the 8- to 16-cell stage, our findings demonstrate a temporal correlation between transcriptional activation and a major rearrangement of chromatin topography in blastomere nuclei.

Three-dimensional maps of all chromosomes in human male fibroblast nuclei and prometaphase rosettes.

Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes-independently of their gene density-were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding.