Eukaryotic genome organization analyzed by electron microscope in situ hybridization

In situ hybridization is a powerful tool for the localization of DNA/RNA sequences in nuclei and chromosomes. The introduction of nonisotopic labelling methodologies in conjunction with fluorescent or enzyme-linked detection have resulted in a dramatic increase in the application of this technique at the light microscope (LM) level and has placed it in a pivotal role in cell biology, development and genetics. Development of equivalent mapping protocols at the EM level offers increased spatial resolution. We have combined the use of nonisotopic probes with invmunogold labelling to investigate eukaryotic genome organization at high resolution.Metaphase chromosomes released from mitotically-arrested cells are deposited on gold EM grids by centrifugation through a sucrose cushion. After fixation (0.1% glutaraldehyde, 20 min) and DNA denaturation, chromosomes are hybridized to cloned probes enzymatically labelled with biotin-dUTP, digoxigenin-dUTP, dinitrophenyl-dUTP or covalently coupled to N-acetoxyacetoaminofluorene. Hybrid sites typically are detected by a two-step antibody incubation and 1-30 nm colloidal gold particles.

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In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

The Journal of Cell Biology ◽

10.1083/jcb.95.2.609 ◽

1982 ◽

Vol 95 (2) ◽

pp. 609-618 ◽

Cited By ~ 85

Author(s):

NJ Hutchison ◽

PR Langer-Safer ◽

DC Ward ◽

BA Hamkalo

Keyword(s):

In Situ Hybridization ◽

Electron Microscope ◽

Satellite Dna ◽

Light Microscope ◽

Test System ◽

Microscope Level ◽

Metaphase Chromosomes ◽

Light Microscope Level ◽

Mouse Satellite Dna

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.

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High-resolution FEISEM detection of DNA centromeric probes in HeLa metaphase chromosomes.

Journal of Histochemistry & Cytochemistry ◽

10.1177/43.4.7897182 ◽

1995 ◽

Vol 43 (4) ◽

pp. 413-419 ◽

Cited By ~ 10

Author(s):

E Rizzi ◽

M Falconi ◽

R Rizzoli ◽

B Baratta ◽

L Manzoli ◽

...

Keyword(s):

In Situ Hybridization ◽

High Resolution ◽

Centromeric Region ◽

Secondary Electron Emission ◽

Dna Probes ◽

Dna Denaturation ◽

Metaphase Chromosomes ◽

Chromatin Arrangement ◽

Fiber Organization

HeLa metaphase chromosome spreads were hybridized with centromeric biotinylated DNA probes and detected with gold-conjugated anti-biotin antibodies. Chromosomes were observed by an in-lens field emission scanning electron microscope (FEISEM), which permits detection of biological samples without any coating. DNA probes were well localized in the centromeric region of chromosomes and there was clear discrimination between 10 nm fibers that hybridized to DNA probes and those that did not hybridize. This approach shows that in situ hybridization can be directly visualized at the FEISEM level by evaluating only secondary electron emission, which allows physical localization of the hybridized probe with high resolution so that backscatter detection represents only a control. Because chromosomes maintain the 10-nm fiber organization after in situ hybridization procedures, our data suggest that this fiber represents the lowest order of chromatin arrangement that permits transitory DNA denaturation.

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DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122885 ◽

1992 ◽

Vol 50 (1) ◽

pp. 496-497

Author(s):

Barbara Trask ◽

Susan Allen ◽

Anne Bergmann ◽

Mari Christensen ◽

Anne Fertitta ◽

...

Keyword(s):

In Situ Hybridization ◽

Dna Sequence ◽

Dna Sequences ◽

Dual Band ◽

Nick Translation ◽

Metaphase Chromosomes ◽

Band Pass ◽

Texas Red ◽

Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

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Ultrastructural analysis of the spatial distribution of MRNA

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100123167 ◽

1992 ◽

Vol 50 (1) ◽

pp. 552-553

Author(s):

Gary Bassell ◽

Robert H. Singer

Keyword(s):

Electron Microscopy ◽

Spatial Distribution ◽

In Situ Hybridization ◽

High Resolution ◽

Nucleic Acids ◽

Colloidal Gold ◽

Dna Probe ◽

Nucleotide Analogs ◽

Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

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Lateral Elements Inside Synaptonemal Complex-Like Polycomplexes in ndt80 Mutants of Yeast Bind DNA

Genetics ◽

10.1093/genetics/163.2.539 ◽

2003 ◽

Vol 163 (2) ◽

pp. 539-544 ◽

Cited By ~ 1

Author(s):

Hasanuzzaman Bhuiyan ◽

Gunilla Dahlfors ◽

Karin Schmekel

Keyword(s):

In Situ Hybridization ◽

Electron Microscope ◽

Synaptonemal Complex ◽

Immunoelectron Microscopy ◽

Meiotic Prophase ◽

Lateral Element ◽

Homologous Chromosomes ◽

Meiotic Prophase I ◽

Dna Antibodies

Abstract The synaptonemal complex (SC) keeps the synapsed homologous chromosomes together during pachytene in meiotic prophase I. Structures that resemble stacks of SCs, polycomplexes, are sometimes found before or after pachytene. We have investigated ndt80 mutants of yeast, which arrest in pachytene. SCs appear normal in spread chromosome preparations, but are only occasionally found in intact nuclei examined in the electron microscope. Instead, large polycomplexes occur in almost every ndt80 mutant nucleus. Immunoelectron microscopy using DNA antibodies show strong preferential labeling to the lateral element parts of the polycomplexes. In situ hybridization using chromosome-specific probes confirms that the chromosomes in ndt80 mutants are paired and attached to the SCs. Our results suggest that polycomplexes can be involved in binding of chromosomes and possibly also in synapsis.

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Application of biotinylated oligonucleotide probes to the detection of pituitary hormone mRNA using Northern blot analysis, in situ hybridization at the light- and electron-microscope levels

The Histochemical Journal ◽

10.1007/bf00188075 ◽

1994 ◽

Vol 26 (10) ◽

pp. 771-777 ◽

Cited By ~ 1

Author(s):

Akira Matsuno ◽

Akira Teramoto ◽

Susumu Takekoshi ◽

Hirotoshi Utsunomiya ◽

Yoshitaka Ohsugi ◽

...

Keyword(s):

In Situ Hybridization ◽

Electron Microscope ◽

Northern Blot ◽

Blot Analysis ◽

Northern Blot Analysis ◽

Pituitary Hormone ◽

Oligonucleotide Probes

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Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.

The Journal of Cell Biology ◽

10.1083/jcb.108.6.2343 ◽

1989 ◽

Vol 108 (6) ◽

pp. 2343-2353 ◽

Cited By ~ 105

Author(s):

R H Singer ◽

G L Langevin ◽

J B Lawrence

Keyword(s):

In Situ Hybridization ◽

High Resolution ◽

Colloidal Gold ◽

Messenger Rna ◽

Signal To Noise Ratio ◽

Simultaneous Detection ◽

Gold Particles ◽

Rna Molecules ◽

Nucleic Acid Molecule

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

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Karyotypes and Distribution of Tandem Repeat Sequences in Brassica nigra Determined by Fluorescence in situ Hybridization

Cytogenetic and Genome Research ◽

10.1159/000479179 ◽

2017 ◽

Vol 152 (3) ◽

pp. 158-165 ◽

Cited By ~ 6

Author(s):

Gui-xiang Wang ◽

Qun-yan He ◽

Jiri Macas ◽

Petr Novák ◽

Pavel Neumann ◽

...

Keyword(s):

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Fluorescence Intensity ◽

Tandem Repeat ◽

Brassica Nigra ◽

Metaphase Chromosomes ◽

Repeat Sequences ◽

Tandem Repeat Sequences ◽

Black Mustard

Whole-genome shotgun reads were analyzed to determine the repeat sequence composition in the genome of black mustard, Brassica nigra (L.) Koch. The analysis showed that satellite DNA sequences are very abundant in the black mustard genome. The distribution pattern of 7 new tandem repeats (BnSAT13, BnSAT28, BnSAT68, BnSAT76, BnSAT114, BnSAT180, and BnSAT200) on black mustard chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of BnSAT13 and BnSAT76 provided useful cytogenetic markers; their position and fluorescence intensity allowed for unambiguous identification of all 8 somatic metaphase chromosomes. A karyotype showing the location and fluorescence intensity of these tandem repeat sequences together with the position of rDNAs and centromeric retrotransposons of Brassica (CRB) was constructed. The establishment of the FISH-based karyotype in B. nigra provides valuable information that can be used in detailed analyses of B. nigra accessions and derived allopolyploid Brassica species containing the B genome.

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