Subject(s)
CD55 Antigens/genetics , Gene Transfer Techniques , Myocardium/immunology , Spermatozoa , Animals , Animals, Genetically Modified , Antigens, CD/analysis , Antigens, CD/genetics , CD55 Antigens/analysis , Humans , Immunohistochemistry , Male , Myocardium/cytology , Swine , Transplantation, Heterologous/methodsABSTRACT
This paper demonstrates that (a) differences in the methylation levels of interphase nuclei can be measured on a cell-by-cell basis, (b) the binding sites of beta-satellite DNA and 5-methylcytosine (5MeC)-rich regions can be localised in interphase nuclei and metaphase chromosomes by sequential in situ hybridization and indirect immunolabelling, and (c) quantitative differences in the relative extensions of beta-satellite DNA and anti-5MeC antibody binding areas can also be measured. This goal was achieved by indirect immunolabelling by anti-5MeC antibodies (Reynaud et al.: Cancer Lett. 61:255-262, 1991) of control and 5-azacytidine-treated human cell cultures. A quantitative analysis of the number, total, and mean areas of labelled heterochromatic regions and the optical densities of euchromatin and heterochromatin was performed for the cells on microscope slides. Dedicated software was used to select and measure the areas of cytological interest. In additional experiments, DAPI-stained slides from control cultures were sequentially treated by in situ hybridization with beta-satellite DNA probe and indirect immunofluorescent labelling with anti-5MeC antibodies. Fluorescent signals of probe and antibodies were pseudocoloured and merged on digital images. The relative locations of probe- and antibody-positive areas were analysed on metaphases and nuclei, and their extensions were quantified in interphase nuclei. Our results show that (a) our analysis can successfully detect different levels of DNA methylation within individual nuclei, (b) in metaphase chromosomes the antibody binding sites are mostly coincident with the hybridisation sites, and (c) in interphase nuclei a quite different picture is consistently observed.
Subject(s)
DNA Methylation , Image Processing, Computer-Assisted/methods , Interphase , 5-Methylcytosine , Cell Line , Cell Nucleus/chemistry , Cytosine/analogs & derivatives , Cytosine/analysis , DNA Probes , DNA, Satellite , Female , Fluorescent Antibody Technique, Indirect , Heterochromatin , Humans , In Situ Hybridization, Fluorescence , Lymphocytes , Male , Metaphase , SoftwareABSTRACT
Reciprocal translocation t(11;22)(q23;q11) is of particular interest because the unbalanced offspring of the translocation carriers usually present with a supernumerary derivative chromosome 22. This common unbalanced karyotype is the result of 3:1 chromosome segregation during meiosis. We report the third case of a rare segregation pattern of a paternal 11;22 translocation. The proband's karyotype revealed the presence of a der(11) and two copies of a der(22), i.e. 47,XX,t(11;22)(q23;q11),+der(22) t(11;22)pat. The karyotype is the result of paternal 3:1 segregation after crossing-over involving the derived and the normal chromosome 22, as revealed by chromosome polymorphism analysis. Contrary to the preferential maternal, transmission of this common unbalanced translocation, the data from the literature, including our case, may suggest preferential paternal transmission of this rare type of unbalanced translocation.
Subject(s)
Abnormalities, Multiple/genetics , Chromosome Aberrations/genetics , Chromosomes, Human, Pair 11/genetics , Chromosomes, Human, Pair 22/genetics , Intellectual Disability/genetics , Nondisjunction, Genetic , Spermatogenesis , Translocation, Genetic , Anaphase , Aneuploidy , Chromosome Disorders , Chromosomes, Human, Pair 11/ultrastructure , Chromosomes, Human, Pair 22/ultrastructure , Crossing Over, Genetic , Face/abnormalities , Female , Humans , Infant , Karyotyping , Male , Muscle Hypotonia/genetics , PhenotypeABSTRACT
This work was aimed at studying the effects of the demethylating agent 5-azacytidine (5-azaC) on the constitutive heterochromatin of human chromosomes at the cytological level. Metaphase preparations from peripheral blood lymphocyte and lymphoblastoid cultures obtained by standard methods were treated with the agent. Labelling of the heterochromatic regions was achieved by the indirect immunostaining method using anti-5-methylcytosine (5MeC) monoclonal antibodies and peroxidase-tagged second antibodies. 4-Chloro-1-naphthol (4C1N) was used as the substrate. The rate of methylation of individual chromosomes or chromosome groups was measured as the frequency of binding of anti-5MeC antibodies to specific chromosome regions. The following results were obtained: (i) in control cultures high intra- and interindividual variability in the binding frequencies of anti-5MeC antibodies to the short arm region of the acrocentrics was observed; (ii) 5-azaC consistently affects the methylation status of the constitutive heterochromatin; (iii) preferential demethylation occurs in the heterochromatic regions of specific chromosomes; (iv) under our experimental conditions the demethylating effect of 5-azaC appears not to be related to the well-known uncoiling effect of this drug.
Subject(s)
Azacitidine/pharmacology , Chromosomes, Human/chemistry , DNA Methylation , Heterochromatin/chemistry , 5-Methylcytosine , Antibodies, Monoclonal , Cells, Cultured , Centromere , Cytosine/analogs & derivatives , Cytosine/analysis , DNA Methylation/drug effects , Humans , Karyotyping , Lymphocytes , MaleABSTRACT
The rate of ribosomal gene activity was evaluated by silver staining of the Nucleolus Organisers (NOs) in cultured CHO-K1 cells after a 12 h pulse with two demethylating agents (L-ethionine and 5-azacytidine). Silver staining of the NOs was measured every 24 h, from 24 up to 110 h after seeding. The purpose was to test the hypothesis that drug-induced demethylation is associated to heritable modifications of rDNA activity. Ribosomal gene activity was shown to be significantly increased by both agents. The increase persisted throughout the experiments, thereby suggesting the heritability of this epigenetic modification. The analysis of heritable DNA damage or modification is an important task in studying the risk of cancer onset and the mechanisms of cancer induction. In these studies two main results were obtained: (i) heritable DNA variations can be induced by both mutational and epigenetic changes; (ii) the modified end-point was not negatively selected.
Subject(s)
Antimetabolites/toxicity , DNA, Ribosomal/drug effects , Gene Expression Regulation, Neoplastic , Germ-Line Mutation , Mutagens/toxicity , Animals , Azacitidine/metabolism , Azacitidine/toxicity , CHO Cells , Chromosome Deletion , Clone Cells , Cricetinae , DNA, Ribosomal/metabolism , Ethionine/metabolism , Ethionine/toxicity , Methylation/drug effects , Mitotic Index/drug effects , Mutagens/metabolism , Nucleolus Organizer Region/drug effects , Nucleolus Organizer Region/metabolism , Silver StainingABSTRACT
Ribosomal gene activity and levels of DNA methylation were investigated by cytochemical and immunological methods in the nucleolar organizer regions (NORs) of individually recognised acrocentric chromosomes. Mendelian inheritance of ribosomal gene activity in a three generation family was demonstrated, together with consistent behaviour of individual gene clusters in different carriers, even when environmental conditions were changed. For most chromosomes, an inverse relationship between gene activity and the level of DNA methylation was observed. Exceptions were the two chromosomes 15 and chromosomes 13cp and 22p, all being strongly chromomycin-A3-positive in their short arms. These chromosomes bound to anti-5-MeC antibodies with differential frequencies in the different carriers. The possibility of involvement of repetitive GC-rich DNA in this behaviour is discussed.
