ABSTRACT
Design and development of highly sensitive method bioinformatics are important for investigation of casual relationships between epigenetic changes and gene activity. Cell polyploidy may trigger such changes. However, maintaining the balance of gene dosage, polyploidy may provide only a rather weak effect on their expression. Currently, there is no comprehensive and concordant data in regard to ploidy-associated transcriptomic changes. To find out how polypoidy affects gene activity, we have developed an integrative bioinformatic method of pairwise cross-species transcriptome analysis of mammalian tissues with various polyploidy degrees. The main benefit of this approach is its ability to separate species- and tissue-specific noises of evolutionary conserved effects. We demonstrat the application of the method for the analysis of gene modules and protein interactions networks coordinating programs of development, differentiation and pluripotency. The analysis was performed with transcriptomes of polyploid and diploid organs (human and mouse heart and liver). Our data indicate that ploidy-induced genes enrich Gene Ontology (GO) biological processes and KEGG pathways related to development, morphogenesis and stem cells biology (including Hippo, Pi3K, WNT, Hedgehog and TGF-ß pathways) with higher degree than ploidy-inhibited genes. Thas, our data are the first to show that polyploidy may induce and coordinate developmental modules.
Subject(s)
Gene Expression Regulation, Developmental , Gene Regulatory Networks , Genes, Developmental , Morphogenesis/genetics , Pluripotent Stem Cells/metabolism , Transcriptome , Animals , Computational Biology , Epigenesis, Genetic , Gene Expression , Gene Expression Profiling , Gene Ontology , Heart/growth & development , Humans , Liver/growth & development , Mice , Pluripotent Stem Cells/cytology , Polyploidy , Protein Interaction Mapping , Signal TransductionABSTRACT
As it has been shown earlier, the cells of experimental tumours as well as some normal cells of the thymus, spleen and testes show the phenomenon of nuclear achromasia (low affinity for ionic dyes) in cytological preparations. Possible connection between the phenomenon and activation for proliferation was the subject of the study presented. A reliable increase in the number of cells displaying the nuclear achromasia when staining with methylene blue after treatment with phytohaemagglutinin for 30 min as well as appearance of such cells in rat liver regenerates 1 h after partial hepatectomy was substantiated to be a result of the cell activation as tested by the enhanced acridine orange intercalation into their DNA.
Subject(s)
Cell Nucleus/ultrastructure , Staining and Labeling , Acridine Orange , Animals , Cell Division , Hepatectomy , Liver/cytology , Male , Methylene Blue , Rats , Rats, Inbred Strains , Staining and Labeling/methods , Thymus Gland/cytologyABSTRACT
Single-stranded DNA breaks in Zajdela's ascitic hepatoma and transformed hamster fibroblasts were caused by treating alive cells with 1% dimethylsulfoxide for 2 h or 100 micrograms/ml bleomycin for 5 min and tested by alkaline and neutral DNA elutions. Electron microscopy of thin sections revealed decompaction of the loosened approximately 25 nm globules within diffuse chromatin into thin fibrillar mesh while supranucleosomal structure of the compact chromatin remained untouched. The chromatin enhanced its affinity for cationic dyes and contrast agents. It is concluded that the diffuse chromatin possesses torsional stress of DNA superhelicity and its loosened subunits represent a form for its organization. They probably correspond to the functionally active (dynamic) nucleosomes which display destruction under DNA domain relaxation caused by one-strand breaks.
Subject(s)
Chromatin/ultrastructure , DNA Damage , DNA, Neoplasm/ultrastructure , DNA, Single-Stranded/ultrastructure , Liver Neoplasms, Experimental/ultrastructure , Animals , Bleomycin/pharmacology , Cells, Cultured , Chromatin/drug effects , Chromatin/metabolism , Cricetinae , DNA, Neoplasm/drug effects , DNA, Neoplasm/metabolism , DNA, Single-Stranded/drug effects , DNA, Single-Stranded/metabolism , Dimethyl Sulfoxide/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Histocytochemistry , Liver Neoplasms, Experimental/metabolism , Microscopy, Electron , Tumor Cells, CulturedABSTRACT
Tumour cell nuclei display achromasia in smears relative to methylene blue and to other low-affinitive ionic dyes. In the course of the smear air-drying the tumour DNA undergoes conformational changes as evidenced by enhanced ethidium and monomer acridine orange uptake and increased sensitivity to DNAse I hydrolysis. Very mild treatment with bleomycin prevents nuclear achromasia with methylene blue in tumour cells. It is concluded that the phenomenon of nuclear achromasia of tumour cells is due to the properties of secondary structure of their DNA.
Subject(s)
Chromatin/ultrastructure , Fibrosarcoma/ultrastructure , Lung Neoplasms/ultrastructure , Neoplasms, Experimental/ultrastructure , Animals , Chromatin/drug effects , DNA, Neoplasm/drug effects , DNA, Neoplasm/ultrastructure , Humans , Microscopy, Electron , Nucleic Acid Conformation/drug effects , Protein Conformation/drug effects , Staining and Labeling/methods , Tumor Cells, CulturedABSTRACT
There are two types of DNA-nuclear matrix interactions in animal cells as revealed by the release of DNA from isolated nuclei by three successive gradients: NaCl, LiCl-urea and temperature. Nuclei were treated with dissociating agents while being adsorbed on the Celite columns. "Weak" DNA-matrix interactions which dissociate in 1.5 M LiCl-3 M urea at 2 degrees appear to be sensitive to ethidium bromide and resistant to exogeneous nucleases (DNAase I, DNAase II and micrococcal nuclease), to DNA-damaging agents, including alkylators and gamma-irradiation, and also to psoralen-induced cross-links. "Strong" DNA-matrix interactions proved to be very different. They dissociate in 4 M LiCl-8 M urea at approximately 90 degrees, are very sensitive to DNAase I and other nucleases, slightly sensitive to chemicals and irradiation at doses stimulating single-stranded DNA breaks, but resistant to ethidium bromide. DNA strand separation seems to be necessary prerequisite for DNA release from its "strong" complex with nuclear matrix. A model for the topological link between DNA and the nuclear matrix involved in DNA replication complex is discussed.
Subject(s)
DNA/isolation & purification , Nucleic Acid Conformation , Nucleoproteins/isolation & purification , Animals , Cell Nucleus/analysis , Cells, Cultured , Chromatography , DNA/radiation effects , DNA, Neoplasm/isolation & purification , Diatomaceous Earth , Fibroblasts/analysis , HeLa Cells/analysis , Humans , Hydrolysis , Liver Neoplasms, Experimental/analysis , Methoxsalen/pharmacology , Mice , Nucleic Acid Denaturation , RatsABSTRACT
Thin epoxy sections of formaldehyde-fixed tissue underwent depurinizing hydrolysis with 5N HCl at room temperature for 1 hour, the treatment with an aldehyde blocking agent, 10% sodium bisulfite, at 60 degrees C during 90 min, and staining with 2% aqueous uranyl acetate. Introduction of bisulfite considerably enhanced the contrast of chromatin. Staining of DNA in these conditions is summed up of preferential binding of uranyl cations with phosphates of DNA and with bisulfite anions specifically bound to deoxyribose.
Subject(s)
DNA/metabolism , Liver Neoplasms, Experimental/metabolism , Animals , DNA, Neoplasm/metabolism , Histocytochemistry/methods , RatsABSTRACT
Treatment of semi-thin epoxy sections with 0.1 N Hcl prior to staining with toluidine blue (pH approximately 9.0) ensures contrastic and reproducible image of tissues fixed for electron microscopy.
Subject(s)
Staining and Labeling/methods , Tolonium Chloride , Humans , Microscopy, Electron/methods , Neoplasms/ultrastructureABSTRACT
Direct and indirect methods for electron microscopic detection of DNA are reviewed. Indirect methods based on DNA removal or preferential contrasting of RNP-structures make it possible to define DNA localization only in outline. By means of direct methods it is possible to reveal all three components of DNA-phosphoricacid groups, pseudoaldehyde groups of desoxyribose, nitrogen bases. Within the framework of this classification the reactions suggested so far and the results obtained with them have been discussed. The main difficulty--lack of specificity, low contrast, poor reproduction, damage of ultrastructures.
Subject(s)
DNA, Neoplasm/isolation & purification , Histocytochemistry/methods , Liver Neoplasms/analysis , Animals , Evaluation Studies as Topic , Liver/pathology , Liver/ultrastructure , Liver Neoplasms/ultrastructure , Microscopy, Electron , Neoplasms, Experimental/analysis , Neoplasms, Experimental/ultrastructure , RatsABSTRACT
The author studied the mechanisms and the applicability in histochemistry of the sodium bisulfate treatment with subsequent toluidine and methylene blue staining after Felgen's hydrolysis. Bisulfite treatment proved to increase the reaction intensity 11/2-fold; the stain is bound stoichiometrically. Toludidine blue results in a metachromatic and anisotropic staining of the cell nuclei. The method is recommended as a sensitive test for DNA in cytochemical investigations and for the study of dichroism of the DNA-containing structures.
Subject(s)
DNA/metabolism , Animals , Carcinoma 256, Walker/metabolism , DNA, Neoplasm/metabolism , Histocytochemistry/methods , Liver/metabolism , Methylene Blue , Rats , Spectrum Analysis , Staining and Labeling , Sulfites , Tolonium ChlorideABSTRACT
Some combinations are suggested to reveal cell components in the following sequence: 1) RNA--DNA--total proteins; 2) RNA--DNA--histones; 3) RNA--DNA--histones--total proteins; 4) RNA--DNA--acidic proteins. RNA is stained with pyronin Y (in the mixture with methyl green). DNA--by the Feulgen procedure (with TCA hydrolysis), histones and total proteins--by Alfert--Geschwind's method and acidic proteins--according to the technique of Smetana and Bush. The details of the whole procedure, adopted for formol-fixed smears are described.
Subject(s)
DNA/metabolism , Histocytochemistry/methods , Proteins/metabolism , RNA/metabolism , Histones/metabolismABSTRACT
After hydrolysis with 5N HCl at room temperature Feulgen's reaction either with Schiff's reagent or with some basic dyes instead of it can be carried out on the air-dried smears without any chemical fixation. Both reactions give quantitative results.