Three-dimensional localization of poly(A) RNA and splicing components in the nucleus

The physical distribution of active genes has long been a subject of interest and speculation, however technical limitations have necessitated that it be addressed only by indirect approaches with sometimes contradictory results. However, developments in fluorescence in situ hybridization methodologies allow the position of specific genes and RNAs to be visualized directly within intact cells, thus providing a more direct means to study such questions. To address whether compartmentalization occurs during the production and processing of pre-mRNA in mammalian somatic cells we have recently investigated the distribution of nuclear polyadenylated transcripts which represent approximately 90% of all pre-mRNA. We found that poly(A) RNA forms discrete nuclear “transcript domains” which are specifically positioned with respect to the underlying genome and contain snRNP antigens of the pre-mRNA splicing class. Several lines of evidence indicate a close spatial and temporal linkage between transcription and processing of pol II RNAs (reviewed in 5), therefore, it is possible that poly(A) RNA transcript domains reflect a clustering of active genes at these sites.

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Detection and mapping of interactions between chromatin and the nuclear envelope in drosophila embryos

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140191 ◽

1995 ◽

Vol 53 ◽

pp. 764-765

Author(s):

W.F. Marshall ◽

A.F. Dernburg ◽

B. Harmon ◽

J.W. Sedat

Keyword(s):

Nuclear Envelope ◽

Chromatin Condensation ◽

Three Dimensional ◽

Physiological Role ◽

Nuclear Surface ◽

Intact Cells ◽

Molecular Determinants ◽

Surface Harmonic ◽

Drosophila Embryos

Interactions between chromatin and nuclear envelope (NE) have been implicated in chromatin condensation, gene regulation, nuclear reassembly, and organization of chromosomes within the nucleus. To further investigate the physiological role played by such interactions, it will be necessary to determine which loci specifically interact with the nuclear envelope. This will not only facilitate identification of the molecular determinants of this interaction, but will also allow manipulation of the pattern of chromatin-NE interactions to probe possible functions. We have developed a microscopic approach to detect and map chromatin-NE interactions inside intact cells.Fluorescence in situ hybridization (FISH) is used to localize specific chromosomal regions within the nucleus of Drosophila embryos and anti-lamin immunofluorescence is used to detect the nuclear envelope. Widefield deconvolution microscopy is then used to obtain a three-dimensional image of the sample (Fig. 1). The nuclear surface is represented by a surface-harmonic expansion (Fig 2). A statistical test for association of the FISH spot with the surface is then performed.

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New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis

Cells ◽

10.3390/cells10071819 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1819

Author(s):

Tatyana Karamysheva ◽

Svetlana Romanenko ◽

Alexey Makunin ◽

Marija Rajičić ◽

Alexey Bogdanov ◽

...

Keyword(s):

In Situ Hybridization ◽

Sex Chromosomes ◽

Interphase Nucleus ◽

Spatial Organization ◽

Three Dimensional ◽

Dna Probes ◽

B Chromosomes ◽

Apodemus Flavicollis ◽

Yellow Necked Mouse

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

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Differential expression of the msp130 gene among skeletal lineage cells in the sea urchin embryo: a three dimensional in situ hybridization analysis

Mechanisms of Development ◽

10.1016/0925-4773(92)90079-y ◽

1992 ◽

Vol 37 (3) ◽

pp. 173-184 ◽

Cited By ~ 40

Author(s):

Michael Alan Harkey ◽

Helen R. Whiteley ◽

Arthur H. Whiteley

Keyword(s):

In Situ Hybridization ◽

Differential Expression ◽

Sea Urchin ◽

Three Dimensional ◽

Hybridization Analysis ◽

Sea Urchin Embryo

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Distinct contracted conformations of the Tcra/Tcrd locus during Tcra and Tcrd recombination

Journal of Experimental Medicine ◽

10.1084/jem.20100772 ◽

2010 ◽

Vol 207 (9) ◽

pp. 1835-1841 ◽

Cited By ~ 36

Author(s):

Han-Yu Shih ◽

Michael S. Krangel

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Three Dimensional ◽

Double Negative ◽

Thymocyte Development ◽

Double Positive ◽

Long Distance ◽

Large Pool ◽

Antigen Receptor Loci

Studies have suggested that antigen receptor loci adopt contracted conformations to promote long-distance interactions between gene segments during V(D)J recombination. The Tcra/Tcrd locus is unique because it undergoes highly divergent Tcrd and Tcra recombination programs in CD4−CD8− double negative (DN) and CD4+CD8+ double positive (DP) thymocytes, respectively. Using three-dimensional fluorescence in situ hybridization, we asked whether these divergent recombination programs are supported by distinct conformational states of the Tcra/Tcrd locus. We found that the 3′ portion of the locus is contracted in DN and DP thymocytes but not in B cells. Remarkably, the 5′ portion of the locus is contracted in DN thymocytes but is decontracted in DP thymocytes. We propose that the fully contracted conformation in DN thymocytes allows Tcrd rearrangements involving Vδ gene segments distributed over 1 Mb, whereas the unique 3′-contracted, 5′-decontracted conformation in DP thymocytes biases initial Tcra rearrangements to the most 3′ of the available Vα gene segments. This would maintain a large pool of distal 5′ Vα gene segments for subsequent rounds of recombination. Thus, distinct contracted conformations of the Tcra/Tcrd locus may facilitate a transition from a Tcrd to a Tcra mode of recombination during thymocyte development.

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Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.11.1918926 ◽

1991 ◽

Vol 39 (11) ◽

pp. 1495-1506 ◽

Cited By ~ 22

Author(s):

P M Motte ◽

R Loppes ◽

M Menager ◽

R Deltour

Keyword(s):

Electron Microscopy ◽

In Situ Hybridization ◽

Spatial Organization ◽

Higher Plants ◽

Three Dimensional ◽

Spatial Arrangement ◽

Thin Sections ◽

Cytoplasmic Dna ◽

Immunocytochemical Techniques

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.

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Three-Dimensional Laser-Scanning Confocal Microscopy of In Situ Hybridization in the Skin

American Journal of Dermatopathology ◽

10.1097/00000372-199402000-00009 ◽

1994 ◽

Vol 16 (1) ◽

pp. 44-51 ◽

Cited By ~ 3

Author(s):

Stephen E. Mahoney ◽

Stephen W. Paddock ◽

Louis C. Smith ◽

Dorothy E. Lewis ◽

Madeleine Duvic

Keyword(s):

In Situ Hybridization ◽

Confocal Microscopy ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Confocal Microscopy ◽

Scanning Confocal Microscopy

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Spatial- and temporal-restricted pattern for amelogenin gene expression during mouse molar tooth organogenesis

Development ◽

10.1242/dev.104.1.77 ◽

1988 ◽

Vol 104 (1) ◽

pp. 77-85 ◽

Cited By ~ 2

Author(s):

M.L. Snead ◽

W. Luo ◽

E.C. Lau ◽

H.C. Slavkin

Keyword(s):

Gene Expression ◽

In Situ Hybridization ◽

Gene Transcription ◽

Developmental Stages ◽

Three Dimensional ◽

Molar Tooth ◽

Specific Expression ◽

Amelogenin Gene ◽

Stage Of Development

Position- and time-restricted amelogenin gene transcription was analysed in developing tooth organs using in situ hybridization with asymmetric complementary RNA probes produced from a cDNA specific to the mouse 26 × 10(3) Mr amelogenin. In situ analysis was performed on developmentally staged fetal and neonatal mouse mandibular first (M1) and maxillary first (M1) molar tooth organs using serial sections and three-dimensional reconstruction. Amelogenin mRNA was first detected in a cluster of ameloblasts along one cusp of the M1 molar at the newborn stage of development. In subsequent developmental stages, amelogenin transcripts were detected within foci of ameloblasts lining each of the five cusps comprising the molar crown form. The number of amelogenin transcripts appeared to be position-dependent, being more abundant on one cusp surface while reduced along the opposite surface. Amelogenin gene transcription was found to be bilaterally symmetric between the developing right and left M1 molars, and complementary between the M1 and M1 developing molars; indicating position-restricted gene expression resulting in organ stereoisomerism. The application of in situ hybridization to forming tooth organ geometry provides a novel strategy to define epithelial-mesenchymal signal(s) which are believed to be responsible for organ morphogenesis, as well as for temporal- and spatial-restricted tissue-specific expression of enamel extracellular matrix.

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