Structural and biochemical aspects of liver cell nuclear organization in normal and scalded rats

1984 ◽  
Vol 71 (1) ◽  
pp. 75-93
Author(s):  
L. Sevaljevic ◽  
M. Petrovic ◽  
G. Poznanovic ◽  
A. Skaro ◽  
D. Pantelic
Keyword(s):  
Nuclear Structure ◽  
Thermal Injury ◽  
Nuclear Organization ◽  
Chromatin Condensation ◽  
Metal Chelators ◽  
Nuclear Pores ◽  
Dna And Rna ◽  
Liver Nuclei ◽  
Minimal Residual

The response of rat liver nuclei to thermal injury was studied at the ultrastructural and biochemical levels using nuclei isolated in the presence of either Mg2+ or polyamines and metal chelators. The extent of chromatin condensation, as revealed by electron microscopy, increased in the order: nuclei in situ, Mg-stabilized nuclei, polyamine-stabilized nuclei. In addition, we observed that thermal injury caused an increase in the number of nuclear pores, an enlargement of nucleoli and an accumulation of ribonucleoprotein material. Along with this, greater amounts of protein, DNA and RNA were retained in nuclei from scalded rats. The salt-resistant residue from Mg-stabilized nuclei was a spherical proteinaceous structure of the nuclear matrix, whereas that of the polyamine-stabilized nuclei was amorphous and deprived of three major constituents of the spherical matrix, the 60–70 X 10(3) Mr lamina proteins. However, exposure of the polyamine-stabilized nuclei to Ca2+ and Mg2+ rendered the 60–70 X 10(3) Mr proteins salt-insoluble and thus enabled the extraction of a spherical residual nuclear structure. This structure was highly enriched in DNA, RNA and non-histone proteins and, hence, more like dehistonized nuclei than the minimal residual nuclear structure. It retained 70% of DNA in the controls but virtually all the DNA in scalded rats. This difference was interpreted to reflect activation-related changes in chromatin organization.

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

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.

High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

1995 ◽  
Vol 53 ◽  
pp. 752-753
Author(s):  
B.A. Hamkalo ◽  
S. Narayanswami ◽  
A.P. Kausch
Keyword(s):  
Nucleic Acid ◽  
Interphase Nucleus ◽  
Genome Mapping ◽  
Precise Location ◽  
Electron Microscope Level ◽  
Rna Sequences ◽  
Fundamental Interest ◽  
Whole Cells ◽  
Dna And Rna

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

Effects of Mg2+ on Chromatic Structure in Isolated Chicken Liver Nuclei

1990 ◽  
Vol 48 (3) ◽  
pp. 130-131
Author(s):  
Soichiro Arai ◽  
Yuh H. Nakanishi
Keyword(s):  
Chromatin Structure ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Divalent Cations ◽  
Chromatin Condensation ◽  
Chicken Liver ◽  
Electron Microscopic ◽  
Razor Blade ◽  
Microscopic Studies ◽  
Liver Nuclei

Although many electron microscopic studies on extracted chromatin have provided considerable information on chromatin condensation induced by divalent cations, there is only a little literature available on the effects of divalent cations on chromatin structure in intact nuclei. In the present study, the effects of Mg2+ on chromatin structure in isolated chicken liver nuclei were examined over a wide concentration range of Mg2+ by scanning electron microscopy.Nuclei were prepared from chicken liver by the method of Chauveau et al. with some modifications. The nuclei were suspended in 25 mM triethanolamine chloride buffer (pH7.4) with 1 mM EDTA or in the buffer with concentrations of MgCl2 varying from 1 to 50 mM. After incubation for 1 min at 0°C, glutaraldehyde was added to 1.8% and the nuclei were fixed for 1 h at 4°C. The fixed nuclei were mixed with 15% gelatin solution warmed at about 40°C, and kept at room temperature until the mixture set. The gelatin containing the nuclei was fixed with 2% glutaraldehyde for 2-4 h, and cut into small blocks. The gelatin blocks were conductive-stained with 2% tannic acid and 2% osmium tetroxide, dehydrated in a graded series of ethanol, and freeze-cracked with a razor blade in liquid nitrogen.

Localization and genotyping of canine papillomavirus in canine inverted papillomas

2021 ◽  
pp. 104063872110357
Author(s):  
Margherita Orlandi ◽  
Maurizio Mazzei ◽  
Marta Vascellari ◽  
Erica Melchiotti ◽  
Claudia Zanardello ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Inclusion Bodies ◽  
Animal Species ◽  
Viral Etiology ◽  
Molecular Approaches ◽  
Dna And Rna ◽  
Promising Tool ◽  
First Choice ◽  
Inverted Papillomas

Numerous canine papillomaviruses (CPVs) have been identified (CPV1–23). CPV1, 2, and 6 have been associated with inverted papillomas (IPs). We retrieved 19 IPs from 3 histopathology archives, and evaluated and scored koilocytes, inclusion bodies, giant keratohyalin granules, cytoplasmic pallor, ballooning degeneration, and parakeratosis. IHC targeting major capsid proteins of PV was performed, and CPV genotyping was achieved by PCR testing. Tissue localization of CPV DNA and RNA was studied by chromogenic and RNAscope in situ hybridization (DNA-CISH, RNA-ISH, respectively). IPs were localized to the limbs (50%), trunk (30%), and head (20%), mainly as single nodules (16 of 19). In 15 of 19 cases, immunopositivity was detected within the nuclei in corneal and subcorneal epidermal layers. PCR revealed CPV1 in 11 IPs and CPV2 DNA in 3 IPs. Overall, 14 of 17 cases were positive by both DNA-CISH and RNA-ISH, in accord with PCR results. A histologic score >5 was always obtained in cases in which the viral etiology was demonstrated by IHC, DNA-CISH, and RNA-ISH. IHC and molecular approaches were useful to ascertain the viral etiology of IPs. Although IHC is the first choice for diagnostic purposes, ISH testing allows identification of PV type and the infection phase. RNA-ISH seems a promising tool to deepen our understanding of the pathogenesis of different PV types in animal species.

The bovine sex-determining region Y (Sry) gene and its mRNA transcript are present in Y sperm but not X sperm of bulls

2018 ◽  
Vol 68 (3) ◽  
pp. 321-332 ◽  
Author(s):  
Chun-Mei Han ◽  
Rong Chen ◽  
Tao Li ◽  
Xiao-Li Chen ◽  
Yong-Fu Zheng ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Fish Analysis ◽  
Mrna Transcript ◽  
Rt Pcr ◽  
Sry Gene ◽  
Chain Reaction ◽  
Dna And Rna ◽  
Positive Rate ◽  
Polymerase Chain

AbstractThe aims of this study were to establish whether the sex-determining region Y gene and its mRNA transcript are present in the Y sperm and X sperm of bulls and, if present, determine their cellular localization. Semen was collected from three bulls and sorted by flow cytometry into X- and Y-chromosome populations. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determineSrymRNA expression in X sperm and Y sperm. The presence and localization ofSryDNA and RNA were investigated by fluorescence in situ hybridization (FISH). RT-PCR detected a singleSrytranscript of 142 bp in Y sperm but not in X sperm. In Y sperm, the FISH-positive rates forSryDNA andSryRNA did not differ significantly from the re-analyzed Y sperm purity. In further experiments, there were no significant differences between the FISH-positive rate forSryRNA and the re-analyzed Y sperm purity for X-sorted, Y-sorted, or unsorted sperm. In conclusion, FISH analysis revealed thatSrytranscripts are present at the edges of the sperm heads of Y sperm but are absent from X sperm.

In Situ Hybridization: Detection of DNA and RNA

2003 ◽  
pp. 27-46 ◽  
Author(s):  
Long Jin ◽  
Xiang Qian ◽  
Ricardo V. Lloyd
Keyword(s):  
In Situ Hybridization ◽  
Dna And Rna ◽  
Hybridization Detection

Denaturation of deoxyribonucleic acid in situ effect of formaldehyde.

1975 ◽  
Vol 23 (6) ◽  
pp. 431-438 ◽  
Author(s):  
F Traganos ◽  
Z Darzyndiewicz ◽  
T Sharpless ◽  
M R Melamed
Keyword(s):  
Deoxyribonucleic Acid ◽  
Direct Action ◽  
Chromatin Condensation ◽  
Cross Linking ◽  
Dna Denaturation ◽  
Cell Type ◽  
Dna Interactions ◽  
Flow Through ◽  
Nuclear Deoxyribonucleic Acid

In situ denaturation of nuclear deoxyribonucleic acid (DNA) is studied by use of acridine orange to differentially stain native versus denatured DNA, and a flow-through cytofluorometer for measurements of cell fluorescence. Thermal- or acid-induced DNA denaturation is markedly influenced by formaldehyde. Two mechanisms of the formaldehyde action are distinguished. If cells are exposed to the agent during heating, DNA denaturation is facilitated, most likely by the direct action of formaldehyde as a "passive" denaturing agent on DNA. If cells are pretreated with formaldehyde which is then removed, DNA resistance to denaturation increases, presumably due to chromatin cross-linking. It is believed that both effects occur simultaneously in conventional techniques employing formaldehyde to study DNA in situ, and that the extent of each varies with the temperature and cell type (chromatin condensation). Thus, profiles of DNA denaturation of cells heated with formaldehyde do not represent characteristics of DNA denaturation in situ; DNA denaturation under these conditions is modulated by the reactivity of chromatin components with formaldehyde rather than by DNA interactions with the macromolecules of nuclear mileu.

scholarly journals Detection of viral DNA and RNA by in situ hybridization.

1986 ◽  
Vol 34 (1) ◽  
pp. 33-38 ◽  
Author(s):  
J K McDougall ◽  
D Myerson ◽  
A M Beckmann
Keyword(s):  
Dna Sequences ◽  
Viral Dna ◽  
Rna Sequences ◽  
Hpv Dna ◽  
Dna And Rna ◽  
Common Warts ◽  
Anogenital Region ◽  
Simplex Virus ◽  
Human Viruses

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.

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