RESUMEN
During meiosis, homologous chromosomes exchange genetic material. This exchange or meiotic recombination is mediated by a proteinaceous scaffold known as the Synaptonemal complex (SC). Any defects in its formation produce failures in meiotic recombination, chromosome segregation and meiosis completion. It has been proposed that DNA repair events that will be resolved by crossover between homologous chromosomes are predetermined by the SC. Hence, structural analysis of the organization of the DNA in the SC could shed light on the process of crossover interference. In this work, we employed an ultrastructural DNA staining technique on mouse testis and followed nuclei of pachytene cells. We observed structures organized similarly to the SCs stained with conventional techniques. These structures, presumably the DNA in the SCs, are delineating the edges of both lateral elements and no staining was observed between them. DNA in the LEs resembles two parallel tracks. However, a bubble-like staining pattern in certain regions of the SC was observed. Furthermore, this staining pattern is found in SCs formed between non-homologous chromosomes, in SCs formed between sister chromatids and in SCs without lateral elements, suggesting that this particular organization of the DNA is determined by the synapsis of the chromosomes despite their lack of homology or the presence of partially formed SCs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Meiosis/fisiología , Complejo Sinaptonémico/metabolismo , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromátides/química , Cromátides/metabolismo , Cromátides/ultraestructura , Emparejamiento Cromosómico/fisiología , ADN/química , ADN/ultraestructura , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Estructura Cuaternaria de Proteína , Complejo Sinaptonémico/fisiología , Complejo Sinaptonémico/ultraestructuraRESUMEN
Fanconi anemia (FA), a chromosomal instability syndrome, is caused by inherited pathogenic variants in any of 22 FANC genes, which cooperate in the FA/BRCA pathway. This pathway regulates the repair of DNA interstrand crosslinks (ICLs) through homologous recombination. In FA proper repair of ICLs is impaired and accumulation of toxic DNA double strand breaks occurs. To repair this type of DNA damage, FA cells activate alternative error-prone DNA repair pathways, which may lead to the formation of gross structural chromosome aberrations of which radial figures are the hallmark of FA, and their segregation during cell division are the origin of subsequent aberrations such as translocations, dicentrics and acentric fragments. The deficiency in DNA repair has pleiotropic consequences in the phenotype of patients with FA, including developmental alterations, bone marrow failure and an extreme risk to develop cancer. The mechanisms leading to the physical abnormalities during embryonic development have not been clearly elucidated, however FA has features of premature aging with chronic inflammation mediated by pro-inflammatory cytokines, which results in tissue attrition, selection of malignant clones and cancer onset. Moreover, chromosomal instability and cell death are not exclusive of the somatic compartment, they also affect germinal cells, as evidenced by the infertility observed in patients with FA.
Asunto(s)
Inestabilidad Cromosómica , Reparación del ADN , Anemia de Fanconi/genética , Envejecimiento/genética , Proteína BRCA1/fisiología , Proteína BRCA2/fisiología , Trastornos de Fallo de la Médula Ósea/etiología , Ciclo Celular , Cromátides/ultraestructura , Aberraciones Cromosómicas , Cromosomas Humanos/ultraestructura , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Anemia de Fanconi/complicaciones , Anemia de Fanconi/diagnóstico , Proteína del Grupo de Complementación C de la Anemia de Fanconi/deficiencia , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación C de la Anemia de Fanconi/fisiología , Humanos , Infertilidad/genética , Síndromes Neoplásicos Hereditarios/genética , Fenotipo , Procesamiento Proteico-Postraduccional , UbiquitinaciónRESUMEN
The topographic structure of Giemsa-banded (G-banded) early metaphase human chromosomes adsorbed on glass was analyzed by atomic force microscope using amplitude modulation mode (AM-AFM). Longitudinal height measurements for early metaphasic human chromosomes showed a central ridge that was further characterized by transversal height measurements. The heterochromatic regions displayed a high level of transversal symmetry, while the euchromatic ones presented several peaks across the transversal height measurements. We suggest that this central ridge and symmetry patterns point out a transitional arrangement of the early metaphase chromosome and support evidence for interchromatidal interactions prior to disjunction.
Asunto(s)
Cromátides/metabolismo , Cromosomas Humanos Par 1/metabolismo , Metafase , Adulto , Cromátides/ultraestructura , Bandeo Cromosómico , Cromosomas Humanos Par 1/ultraestructura , Eucromatina/ultraestructura , Femenino , Heterocromatina/ultraestructura , Humanos , MasculinoRESUMEN
In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90-95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.
Asunto(s)
Células CHO/efectos de la radiación , Daño del ADN , Adaptación Fisiológica , Animales , Células CHO/ultraestructura , Cromátides/efectos de la radiación , Cromátides/ultraestructura , Aberraciones Cromosómicas , Rotura Cromosómica , Cromosomas/efectos de la radiación , Cromosomas/ultraestructura , Ensayo Cometa , Cricetinae , Cricetulus , ADN/efectos de la radiación , Reparación del ADN , Relación Dosis-Respuesta en la Radiación , Procesamiento de Imagen Asistido por Computador , Transferencia Lineal de Energía , Microscopía Fluorescente , Tolerancia a RadiaciónRESUMEN
SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.
Asunto(s)
Células CHO/efectos de los fármacos , Células CHO/efectos de la radiación , Rotura Cromosómica , Inhibidores Enzimáticos/toxicidad , Etopósido/toxicidad , Inhibidores de Topoisomerasa II , Cromosoma X/efectos de los fármacos , Cromosoma X/efectos de la radiación , Animales , Células CHO/ultraestructura , Cromátides/efectos de los fármacos , Cromátides/efectos de la radiación , Cromátides/ultraestructura , Aberraciones Cromosómicas , Mapeo Cromosómico , Cricetinae , Cricetulus , ADN/efectos de los fármacos , ADN/efectos de la radiación , Daño del ADN , Femenino , Fase S/efectos de los fármacos , Fase S/efectos de la radiación , Cromosoma X/genética , Cromosoma X/ultraestructuraRESUMEN
Hemipteran chromosomes are holocentric and show regular, special behavior at meiosis. While the autosomes pair at pachytene, have synaptonemal complexes (SCs) and recombination nodules (RNs) and segregate at anaphase I, the sex chromosomes do not form an SC or RNs, divide equationally at anaphase I, and their chromatids segregate at anaphase II. Here we show that this behavior is shared by the X and Y chromosomes of Triatoma infestans and the X(1)X(2)Y chromosomes of Triatoma pallidipennis. As Rec8p is a widely occurring component of meiotic cohesin, involved in meiotic homolog segregation, we used an antibody against Rec8p of Caenorhabditis elegans for immunolocalization in these triatomines. We show that while Rec8p is colocalized with SCs in the autosomes, no Rec8p can be found by immunolabeling in the sex chromosomes at any stage of meiosis. Furthermore, Rec8p labeling is lost from autosomal bivalents prior to metaphase I. In both triatomine species the sex chromosomes conjoin with each other during prophase I, and lack any SC, but they form "fuzzy cores", which are observed with silver staining and with light and electron microscopy during pachytene. Thin, serial sectioning and electron microscopy of spermatocytes at metaphases I and II reveals differential behavior of the sex chromosomes. At metaphase I the sex chromosomes form separate entities, each surrounded by a membranous sheath. On the other hand, at metaphase II the sex chromatids are closely tied and surrounded by a shared membranous sheath. The peculiar features of meiosis in these hemipterans suggest that they depart from the standard meiotic mechanisms proposed for other organisms.
Asunto(s)
Fosfoproteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Cromosomas Sexuales/ultraestructura , Espermatocitos/ultraestructura , Triatoma/genética , Animales , Cromátides/genética , Cromátides/ultraestructura , Femenino , Inmunohistoquímica , Indoles , Masculino , Meiosis , Ratones , Microscopía Electrónica , Espermatocitos/metabolismo , Huso Acromático , Complejo Sinaptonémico/ultraestructura , Triatoma/ultraestructuraRESUMEN
Chromatin buds (CHB), broken eggs, or budding cell nuclei are structures similar to micronuclei (MN) in shape, structure, and size, which are linked to the main nuclei of cells by a thread or stalks of chromatin. They have been observed in numerous cell types and there are reports of their existence relating them with MN or with genotoxic events. However, there is no systematic study reporting their frequency and no experiment has been done to ascertain whether they are really induced by genotoxins. Furthermore, they have been discarded as genotoxic events with the argument that they are not formed in dividing cells. Studies are presented here that indicate that CHB can be considered as genotoxic events and that their origin is comparable to that of MN. Bromodeoxyuridine (BrdU) was used to label proliferating lymphocytes, which were later identified by means of an immunohistochemical method, using the H2O2-DAB stain. The results show that CHB are consistently formed where MN are seen. CHB were induced by the clastogen mitomycin C (MMC) as well as by the aneuploidogen colcemid, with frequencies similar to MN in both cases, and to multinucleated cells in the case of colcemid. CHB occur in lymphocytes of smokers with frequencies similar to those of MN, and we found that the infection with Taenia solium metacestodes induced a comparable increase of both MN and CHB frequency in lymphocytes from pigs.
Asunto(s)
Cromátides/ultraestructura , Demecolcina/toxicidad , Micronúcleos con Defecto Cromosómico , Mitomicina/toxicidad , Mutágenos/toxicidad , Aneuploidia , Animales , Humanos , Porcinos , Taenia/patogenicidadRESUMEN
The possibility that Ureaplasma urealyticum might play an important role in human infertility was first raised more than 20 years ago, but this association remains speculative. Considering the hypothesis that the pathogenicity of Ureaplasma urealyticum may depend on its serotypes, the clastogenic effcts of different strains of Ureaplasma urealyticum, at concentrations of 10(3) CCU (color changing units)/ml, 10(4) CCU/ml and 10(5) CCU/ml, were evaluated in vitro in short-term cultures of human lyphocytes. Total or partial mitotic inhibition was produced by Ureaplasma urealyticum serotypes 2,3 and 10 independent of the concentration (10(3) CCU/ml, 10(4) CCU/ml or 10 (5) CCU/ml) of the microorganisms employed. In contrast, the clastogenic effects observed with serotypes 1,7 and 12 varied according to the concentration employed in the test. Mitotic alterations were observed in Ureaplasma urealyticum serotypes 5,6,7,8,9,11 and 12. Chromatid gaps (53.0 percent) and chromatid breaks (13.9 percent) were the most frequent types of alterations observed. The results of this in vitro assay demonstrated that the clastogenic effects varied with the Ureaplasma urealyticum serotypes evaluated.
Asunto(s)
Humanos , Cromátides/ultraestructura , Cromosomas Humanos/microbiología , Cromosomas Humanos/ultraestructura , Linfocitos/microbiología , Mitosis/genética , Mutágenos/efectos adversos , Ureaplasma urealyticum/patogenicidad , Cromosomas Humanos/genética , Ureaplasma urealyticum/genéticaRESUMEN
Infection with Trypanosoma cruzi is known to induce the division of peritoneal macrophages in BALB/c mice. We have demonstrated, by cytogenetic analysis, that accessory DNA elements are associated with the metaphase macrophage chromosomes of such infected macrophages. The identification of these accessory DNA elements with T. cruzi DNA is strongly supported by the association of 3H-label with some chromatids in macrophages previously infected with T. cruzi which had been labelled with 3H-methyl-thymidine. The karyotyping consistently showed preferential associations of T. cruzi DNA with chromosomes 3, 6 and 11. A conclusive demonstration of the parasite origin of the integrated DNA came from fluorescein in situ hybridization studies using specific parasite DNAs as probes. In order to determine the identity of the inserted DNA and to investigate the nature of the integration mechanism, Southern blot analyses were performed on DNA extracted from both uninfected and infected (but parasite-free) macrophages. Hybridizations of BamHI, EcoRI and TaqI digests of DNA from T. cruzi-infected host cells all revealed the presence of a 1.7-kb DNA fragment when probed with kDNA. The covalent association of kDNA with that of the host was confirmed by the fact that AluI and Hinf-I digests of DNA from infected host cells produced a number of bands, in a size range of 0.8-3.6 kb, which hybridized with kDNA minicircles. None of these bands was found in DNA purified from cell-free preparations of the parasite and thus it must be concluded that they represent insertion fragments between parasite and host cell DNA. These results strongly suggest that kDNA minicircles from T. cruzi have been integrated into the genome of the host cell following infection.