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Identification of an interchromosomal compartment by polymerization of nuclear-targeted vimentin
A number of structural and functional subnuclear compartments have been described, including regions
exclusive of chromosomes previously hypothesized to form
a reactive nuclear space. We have now explored this
accessible nuclear space and interchromosomal
nucleoplasmic domains experimentally using Xenopus
vimentin engineered to contain a nuclear localization signal
(NLS-vimentin). In stably transfected human cells
incubated at 37°C, the NLS-vimentin formed a restricted
number of intranuclear speckles. At 28°C, the optimal
temperature for assembly of the amphibian protein, NLSvimentin
progressively extended with time out from the
speckles into strictly orientated intranuclear filamentous
arrays. This enabled us to observe the development of a
system of interconnecting channel-like areas. Quantitative
analysis based on 3-D imaging microscopy revealed that
these arrays were localized almost exclusively outside of
chromosome territories. During mitosis the filaments
disassembled and dispersed throughout the cytoplasm,
while in anaphase-telophase the vimentin was recruited
back into the nucleus and reassembled into filaments at the
chromosome surfaces, in distributions virtually identical to
those observed in the previous interphase. The filaments
also colocalized with specific nuclear RNAs, coiled bodies
and PML bodies, all situated outside of chromosome
territories, thereby interlinking these structures. This
strongly implies that these nuclear entities coexist in the
same interconnected nuclear compartment. The
assembling NLS-vimentin is restricted to and can be used
to delineate, at least in part, the formerly proposed
reticular interchromosomal domain compartment (ICD).
The properties of NLS-vimentin make it an excellent tool
for performing structural and functional studies on this
compartment
Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics
DNA libraries from sorted human gonosomes were used selectively to stain the X and Y chromosomes in normal and aberrant cultured human cells by chromosomal in situ suppression (CISS-) hybridization. The entire X chromosome was stained in metaphase spreads. Interphase chromosome domains of both the active and inactive X were clearly delineated. CISS-hybridization of the Y chromosome resulted in the specific decoration of the euchromatic part (Ypter-q11), whereas the heterochromatic part (Yq12) remained unlabeled. The stained part of the Y chromosome formed a compact domain in interphase nuclei. This approach was applied to amniotic fluid cells containing a ring chromosome of unknown origin (47,XY; +r). The ring chromosome was not stained by library probes from the gonosomes, thereby suggesting its autosomal origin. The sensitivity of CISS-hybridization was demonstrated by the detection of small translocations and fragments in human lymphocyte metaphase spreads after irradiation with 60Co-gamma-rays. Lymphocyte cultures from two XX-males were investigated by CISS-hybridization with Y-library probes. In both cases, metaphase spreads demonstrated a translocation of Yp-material to the short arm of an X chromosome. The translocated Y-material could also be demonstrated directly in interphase nuclei. CISS-hybridization of autosomes 7 and 13 was used for prenatal diagnosis in a case with a known balanced translocation t(7;13) in the father. The same translocation was observed in amniotic fluid cells from the fetus. Specific staining of the chromosomes involved in such translocations will be particularly important, in the future, in cases that cannot be solved reliably by conventional chromosome banding alone
Working poverty is a widespread but under-analyzed and poorly-measured problem in the US
Data and statistics are integral to policymakers in government trying to tackle problems such as working poverty. And yet, estimates of the proportion of working poor in the US vary from 2 to 19 percent. In new research, Brian C. Thiede, Daniel T. Lichter, and Scott R. Sanders seek to explain the variation in statistics around those in working poverty. They write that estimates about the magnitude of working poverty, and on its incidence among racial and ethnic groups can be sensitive to the often technical choices, often based on assumptions about how people get into poverty, made by those who are doing the estimating
A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones
The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discusse
Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries
The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescencein situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocal laser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional network-like compartment, termed the interchromosome domain (ICD) compartment, in which transcription and splicing of mRNA occurs
Planned Reduction in Electrical Energy Use in Nashville - Davidson County, Tennessee: A Preliminary Assessment
An assessment was carried out of the impacts of the various alternative strategies designed to reduce the rate of electrical energy use in the Nashville-Davidson County area, in the light of a potential crisis in supply. Seven strategies were identified among the major categories of voluntary reduction, price regulation, and mandatory reduction. Thirty-three sub-sectors were identified among residential, commercial and industrial users, and the consequences of imposing the strategies were assessed using a cross-impact matrix. The value of the methodology as an aid to public policy formulation lies in its possible extension to allow direct participation of various affected publics
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