RESUMEN
Classical observations have shown that during the interphase the chromosomal DNA of metazoans is organized in supercoiled loops attached to a compartment known as the nuclear matrix (NM). Fragments of chromosomal DNA able to bind the isolated NM in vitro are known as matrix associated/attachment/addressed regions or MARs. No specific consensus sequence or motif has been found that may constitute a universal, defining feature of MARs. On the other hand, high-salt resistant DNA-NM interactions in situ define true DNA loop anchorage regions or LARs, that might correspond to a subset of the potential MARs but are not necessarily identical to MARs characterized in vitro, since there are several examples of MARs able to bind the NM in vitro but which are not actually bound to the NM in situ. In the present work we assayed the capacity of two LARs, as well as of shorter fragments within such LARs, for binding to the NM in vitro. Paradoxically the isolated (≈2 kb) LARs cannot bind to the NM in vitro while their shorter (≈300 pb) sub-fragments and other non-related but equally short DNA fragments, bind to the NM in a high-salt resistant fashion. Our results suggest that the ability of a given DNA fragment for binding to the NM in vitro primarily depends on the length of the fragment, suggesting that binding to the NM is modulated by the local topology of the DNA fragment in suspension that it is known to depend on the DNA length. J. Cell. Biochem. 118: 4487-4497, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
ADN/química , Hepatocitos/química , Regiones de Fijación a la Matriz , Matriz Nuclear/química , Animales , ADN/metabolismo , Hepatocitos/metabolismo , Masculino , Matriz Nuclear/metabolismo , Ratas , Ratas WistarRESUMEN
Studies of chromatin extensibility have revealed the flow of chromatin and DNA from cell nuclei and chromosomes in response to gravity or mechanical stretch following lysis by hypertonic saline and detergent solutions. Since this phenomenon was first reported, the technical methods by which extended chromatin fibers (ECFs) may be analyzed have been improved. These methods include topochemical assays, fluorescence in situ hybridization, immunofluorescence, electron microscopy, and polarization microscopy. Chromatin and DNA "halos" also have been studied in materials subjected to lysis, especially in a horizontal position or after cytocentrifugation. The analysis of ECF formation is useful not only as a tool for detecting the positioning of certain DNA signals on chromatin filaments, but also for describing diverse DNA-protein associations that may be related to varying transcriptional activities and chromatin supraorganization. A brief review of the methods and applications of ECF formation is presented here. We focus on light microscopy studies of ECF formation in mouse hepatocytes under different chromatin supraorganization and physiological conditions and in sperm cells with different DNA-protein complexes.
Asunto(s)
Cromatina/química , Cromosomas/química , ADN/química , Matriz Nuclear/química , Animales , Birrefringencia , Núcleo Celular/química , Cromatina/metabolismo , Cromosomas/metabolismo , ADN/análisis , ADN/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Hepatocitos/citología , Hibridación Fluorescente in Situ , Masculino , Ratones , Microscopía Electrónica , Matriz Nuclear/metabolismo , Espermatozoides/citologíaRESUMEN
In the mammalian liver the quiescent primary hepatocytes preserve a proliferating potential in vivo, yet natural aging correlates with loss of proliferating potential and progression towards terminal differentiation of the hepatocytes. Thus aged, terminally-differentiated hepatocytes may survive in a de facto post-mitotic state, similarly to early post-mitotic cells, like neurons, suggesting that there might be a common factor linking both cellular states. In the interphase of metazoan cells the nuclear DNA is organized in supercoiled loops anchored to a proteinaceous substructure known as the nuclear matrix (NM). The DNA-NM interactions define a higher-order structure in the cell nucleus (NHOS). Natural aging of the rat liver correlates with a progressive strengthening of the NM framework and the stabilization of the DNA-NM interactions in the hepatocytes indicating that the NHOS becomes highly stable with age. We compared the NHOS of post-mitotic rat neurons with that of aged rat hepatocytes. Our results indicate that a very stable NHOS is a common feature of both aged and post-mitotic cells in vivo.
Asunto(s)
Envejecimiento/fisiología , Núcleo Celular , ADN , Matriz Nuclear , Animales , Núcleo Celular/química , Núcleo Celular/metabolismo , ADN/química , ADN/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Masculino , Matriz Nuclear/química , Matriz Nuclear/metabolismo , Conformación de Ácido Nucleico , Ratas , Ratas WistarRESUMEN
Organophosphorus (OP) pesticides, widely used in agriculture and pest control, are associated with male reproductive effects, including sperm chromatin alterations, but the mechanisms underlying these effects are unknown. The main toxic action of OP is related to phosphorylation of proteins. Chemical alterations in sperm nuclear proteins (protamines), which pack DNA during the last steps of spermatogenesis, contribute to male reproductive toxicity. Therefore, in the present study, we tested the ability of diazinon (DZN), an OP compound, to alter sperm chromatin by phosphorylating nuclear protamines. Mice were injected with a single dose of DZN (8.12 mg/kg, i.p.), and killed 8 and 15 days after treatment. Quality of sperm from epididymis and vas deferens was evaluated through standard methods and chromatin condensation by flow cytometry (DNA Fragmented Index parameters: DFI and DFI%) and fluorescence microscopy using chromomycin-A(3) (CMA(3)). Increases in DFI (15%), DFI% (4.5-fold), and CMA(3) (2-fold) were observed only at 8 days post-treatment, indicating an alteration in sperm chromatin condensation and DNA damage during late spermatid differentiation. In addition, an increase of phosphorous content (approximately 50%) in protamines, especially in the phosphoserine content (approximately 73%), was found at 8 days post-treatment. Sperm viability, motility, and morphology showed significant alterations at this time. These data strongly suggest that spermatozoa exposed during the late steps of maturation were the targets of DZN exposure. The correlation observed between the phosphorous content in nuclear protamines with DFI%, DFI, and CMA(3) provides evidence that phosphorylation of nuclear protamines is involved in the OP effects on sperm chromatin.
Asunto(s)
Cromatina/efectos de los fármacos , Cromatina/metabolismo , Diazinón/efectos adversos , Espermatozoides/anomalías , Espermatozoides/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cromatina/química , Cromomicina A3 , ADN/efectos de los fármacos , ADN/metabolismo , Fragmentación del ADN/efectos de los fármacos , Diazinón/administración & dosificación , Diazinón/química , Evaluación Preclínica de Medicamentos/métodos , Citometría de Flujo/métodos , Inyecciones Intraperitoneales , Sustancias Intercalantes/efectos adversos , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Masculino , México , Ratones , Ratones Endogámicos , Microscopía Fluorescente/métodos , Matriz Nuclear/química , Matriz Nuclear/efectos de los fármacos , Matriz Nuclear/metabolismo , Fosforilación/efectos de los fármacos , Fosfotirosina/química , Protaminas/química , Protaminas/efectos de los fármacos , Protaminas/metabolismo , Motilidad Espermática/efectos de los fármacos , Motilidad Espermática/fisiología , Espermatozoides/química , Factores de Tiempo , Pruebas de Toxicidad/métodosRESUMEN
Nuclear DNA of higher eukaryotes is organized in supercoiled loops anchored to a proteinaceous substructure commonly known as the nuclear matrix. Current evidence suggests that important processes of nuclear physiology, such as replication, transcription, and processing of primary transcripts, take place at macromolecular complexes located at discrete, well-defined sites upon the nuclear matrix. A number of authors have reported that actively transcribed genes are closely associated with the nuclear matrix. The topological relationship between the gene sequences located in the DNA loops and the nuclear matrix appears to be very important for appropriate nuclear physiology. Here, we describe a polymerase chain reaction-based method for directly mapping any DNA sequence position relative to the nuclear matrix that avoids the problem posed by DNA fragments nonspecifically bound to the nuclear matrix, without the need of purifying the specifically nuclear matrix-bound DNA.
Asunto(s)
Mapeo Cromosómico/métodos , ADN/análisis , Matriz Nuclear/química , Reacción en Cadena de la Polimerasa/métodos , Actinas/genética , Adrenoleucodistrofia/genética , Animales , Secuencia de Bases , ADN/química , Cartilla de ADN/genética , Desoxirribonucleasa I/metabolismo , Globinas/genética , Células HeLa , Humanos , Masculino , Conformación de Ácido Nucleico , Ratas , Ratas Wistar , Moldes GenéticosRESUMEN
In the present report we show the distribution of multiple tubulin isoforms in Trichomonas vaginalis and Tritrichomonas foetus, flagellated parasitic protists of the urogenital tracts of human and cattle, respectively, using immunofluorescence and immunoelectron microscopy. We used several monoclonal and polyclonal anti-tubulin antibodies from different sources and recognizing variant tubulin isoforms. Our results demonstrate that: (1) there is a heterogeneous distribution of the different tubulin isoforms in the main microtubular cell structures, such as axostyle, flagella, basal bodies, and mitotic spindle, (2) the axostyle-pelta junction is a structure with high affinity for glutamylated tubulin antibodies in T. foetus, (3) the spindle labeling is positive to anti-glutamylated tubulin and anti-alpha-tubulin (TAT1 and purchased from Amersham) antibodies in T. vaginalis but it is negative in T. foetus, (4) the nuclear matrix and the cytosol presented positive reaction using glutamylated and TAT1 (anti-alpha-tubulin) antibodies only in T. vaginalis, and (5) the Golgi complex exhibited staining using the glutamylated tubulin antibody. The present data corroborate with the idea of the existence of a heterogeneous population of microtubules in these protists and of a subset of intracytoplasmic microtubules. Microtubule diversity may reflect distinct tubulins, diverse microtubule-associated proteins, or a combination of both.
Asunto(s)
Procesamiento Proteico-Postraduccional , Tritrichomonas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Citosol/química , Inmunohistoquímica/métodos , Matriz Nuclear/química , Matriz Nuclear/ultraestructura , Isoformas de Proteínas/química , Tritrichomonas/química , Tritrichomonas/ultraestructura , Tritrichomonas foetus/química , Tritrichomonas foetus/metabolismo , Tritrichomonas foetus/ultraestructura , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructuraRESUMEN
While much evidence indicates a high degree of spatial organization in the nucleus, the underlying molecular structures that support it remain poorly characterized. By extracting with high concentrations of RNase A in a modification of the sequential extraction protocol of Penman, we have identified a novel intranuclear network in the mouse lymphoma cell line, EL-4. Micrographs of embedment-free sections of extracted cells reveal anastomosing filaments of two different diameters: 3-5 nm and 8-10 nm. The 3-5-nm filaments are interconnected in many junctions and appear to blend smoothly into each other. The 8-10-nm fibers frequently split into two 3-5-nm filaments. Some 3-5-nm fibers appear to be connected at 90 degrees angles with the 8-10-nm fibers. All junctions are smooth with no apparent junction protein. Flow cytometric analysis of RNase A- (and DNase I-) extracted nuclear matrices indicates that they do not contain significant amounts of protein that react with anti-actin and anti-vimentin monoclonal antibodies. Extraction of EL-4 nuclear matrices with high salt does not reveal 8-10-nm core filaments described after similar treatment of tumor cell lines of cervical and mammary origin. The novel characteristics of the core filaments in EL-4 lymphoma cells may reflect cell-type specificity of the nuclear matrix.