RESUMEN
Pulmonary tuberculosis is classified as a disease with a genetic predisposition, and therefore, as with other pathologies related to this group of diseases, by pulmonary tuberculosis, special importance is given to finding those markers that enable early identification of risk groups, such as skrinnig in general population and relatives of patients with tuberculosis, which in turn can provide the basis for preventive measures. One of this markers is the level of genome stability. The aim of this study was a comparative evaluation of the functional parameters of the genome variability in patients with sensitive form of pulmonary tuberculosis before and after treatment, and the possibility of its correction with anti-stress peptide bioregulator - epitalon. The studies were conducted using short-term mitoge -stimulated cell cultures of TB patients, before and after treatment. As an indicator of genome stability has been studied the frequency of structural and numerical chromosome aberrations and fragile sites. It is shown, that in intact cultures from patients with pulmonary tuberculosis, before treatment was significantly higher level of frequency of cells with structural chromosome aberrations, that still retained after the treatment. As for epithalon, it appears that was shown a pronounced protective effect after treatment, on the test of chromosome aberrations, by reducing both overall mean frequency aberrant cells and indicators for all individuals. In the study of fragility of chromosomes in patients with primary tuberculosis was found, that in intact cultures, the proportion of cells with chromosomal fragile sites was higher than in control group of healthy individuals, befor and after treatment. High frequency of chromosome fragility persisted by treatment with peptide bioregulator in both cases - before and after treatment. It is suggested that the identified patterns can be correlated with a high incidence of re- TB.
Asunto(s)
Aberraciones Cromosómicas/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Oligopéptidos/administración & dosificación , Tuberculosis Pulmonar/tratamiento farmacológico , Antituberculosos/administración & dosificación , Sitios Frágiles del Cromosoma/efectos de los fármacos , Predisposición Genética a la Enfermedad , Humanos , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/patologíaRESUMEN
Mammalian common fragile sites are loci of frequent chromosome breakage and putative recombination hotspots. Here, we utilized Replication Slow Zones (RSZs), a budding yeast homolog of the mammalian common fragile sites, to examine recombination activities at these loci. We found that rates of URA3 inactivation of a hisG-URA3-hisG reporter at RSZ and non-RSZ loci were comparable under all conditions tested, including those that specifically promote chromosome breakage at RSZs (hydroxyurea [HU], mec1Δ sml1Δ, and high temperature), and those that suppress it (sml1Δ and rrm3Δ). These observations indicate that RSZs are not recombination hotspots and that chromosome fragility and recombination activity can be uncoupled. Results confirmed recombinogenic effects of HU, mec1Δ sml1Δ, and rrm3Δ and identified temperature as a regulator of mitotic recombination. We also found that these conditions altered the nature of recombination outcomes, leading to a significant increase in the frequency of URA3 inactivation via loss of heterozygosity (LOH), the type of genetic alteration involved in cancer development. Further analyses revealed that the increase was likely due to down regulation of intrachromatid and intersister (IC/IS) bias in mitotic recombination, and that RSZs exhibited greater sensitivity to HU dependent loss of IC/IS bias than non RSZ loci. These observations suggest that recombinogenic conditions contribute to genome rearrangements not only by increasing the overall recombination activity, but also by altering the nature of recombination outcomes by their effects on recombination partner choice. Similarly, fragile sites may contribute to cancer more frequently than non-fragile loci due their enhanced sensitivity to certain conditions that down-regulate the IC/IS bias rather than intrinsically higher rates of recombination.
Asunto(s)
Sitios Frágiles del Cromosoma/genética , Replicación del ADN/genética , Mitosis/genética , Rotura Cromosómica/efectos de los fármacos , Sitios Frágiles del Cromosoma/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Hidroxiurea/farmacología , Mutación , Recombinación Genética/efectos de los fármacos , Recombinación Genética/genética , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Genomic rearrangements at the fragile site FRA1E may disrupt the dihydropyrimidine dehydrogenase gene (DPYD) which is involved in 5-fluorouracil (5-FU) catabolism. In triple-negative breast cancer (TNBC), a subtype of breast cancer frequently deficient in DNA repair, we have investigated the susceptibility to acquire copy number variations (CNVs) in DPYD and evaluated their impact on standard adjuvant treatment. METHODS: DPYD CNVs were analysed in 106 TNBC tumour specimens using multiplex ligation-dependent probe amplification (MLPA) analysis. Dihydropyrimidine dehydrogenase (DPD) expression was determined by immunohistochemistry in 146 tumour tissues. RESULTS: In TNBC, we detected 43 (41%) tumour specimens with genomic deletions and/or duplications within DPYD which were associated with higher histological grade (P=0.006) and with rearrangements in the DNA repair gene BRCA1 (P=0.007). Immunohistochemical analysis revealed low, moderate and high DPD expression in 64%, 29% and 7% of all TNBCs, and in 40%, 53% and 7% of TNBCs with DPYD CNVs, respectively. Irrespective of DPD protein levels, the presence of CNVs was significantly related to longer time to progression in patients who had received 5-FU- and/or anthracycline-based polychemotherapy (hazard ratio=0.26 (95% CI: 0.07-0.91), log-rank P=0.023; adjusted for tumour stage: P=0.037). CONCLUSION: Genomic rearrangements in DPYD, rather than aberrant DPD protein levels, reflect a distinct tumour profile associated with prolonged time to progression upon first-line chemotherapy in TNBC.
Asunto(s)
Variaciones en el Número de Copia de ADN , Dihidrouracilo Deshidrogenasa (NADP)/genética , Recurrencia Local de Neoplasia/genética , Neoplasias de la Mama Triple Negativas/genética , Antimetabolitos Antineoplásicos/uso terapéutico , Proteína BRCA1/genética , Sitios Frágiles del Cromosoma/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Fluorouracilo/uso terapéutico , Eliminación de Gen , Duplicación de Gen/efectos de los fármacos , Duplicación de Gen/genética , Reordenamiento Génico/efectos de los fármacos , Humanos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/enzimología , Recurrencia Local de Neoplasia/patología , Pronóstico , Radiografía , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/enzimologíaRESUMEN
Our previous study demonstrated that 45S ribosomal DNA (45S rDNA) clusters were chromosome fragile sites expressed spontaneously in Lolium. In this study, fragile phenotypes of 45S rDNA were observed under aphidicolin (APH) incubation in several plant species. Further actinomycin D (ActD) treatment showed that transcriptional stress might interfere with chromatin packaging, resulting in 45S rDNA fragile expression. These data identified 45S rDNA sites as replication-dependent as well as transcription-dependent fragile sites in plants. In the presence of ActD, a dramatic switch to an open chromatin conformation and accumulated incomplete 5' end of the external transcribed spacer (5'ETS) transcripts were observed, accompanied by decreased DNA methylation, decreased levels of histone H3, and increased histone acetylation and levels of H3K4me2, suggesting that these epigenetic alterations are associated with failure of 45S rDNA condensation. Furthermore, the finding that γ-H2AX was accumulated at 45S rDNA sites following ActD treatment suggested that the DNA damage signaling pathway was associated with the appearance of 45S rDNA fragile phenotypes. Our data provide a link between 45S rDNA transcription and chromatin-packaging defects and open the door for further identifying the molecular mechanism involved.
Asunto(s)
Sitios Frágiles del Cromosoma/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Lolium/genética , ARN Ribosómico/genética , Afidicolina/administración & dosificación , Cromatina/genética , Sitios Frágiles del Cromosoma/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Dactinomicina/administración & dosificación , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genoma de Planta/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genéticaRESUMEN
Human exposure to metals is of increasing concern due to the well-documented toxic and carcinogenic effects of metals and metal compounds, and the rising environmental levels due to industrial processes and pollution. It has been reported that metals can be genotoxic by several modes of action including generation of reactive oxygen species and inhibition of DNA repair. The aim of this study was to evaluate microsatellite instability (MSI) in three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located within three common fragile sites in the genome of the laboratory rat (6q21, 9q32-9q33 and 15p14) exposed to acute and chronic doses of a metal salt (lead acetate trihydrate) and a metalloid oxide (arsenic trioxide). In the acute and sub-chronic studies with the two compounds, MSI was observed in the three loci as deletions or additions of microsatellite repeats. The percentages of MSI were 36.4% and 42.1% for lead acetate and arsenic trioxide, respectively. Results of the present work indicate that the microsatellites located within fragile sites provide a convenient assay system to detect changes in DNA sequences resulting from exposure to genotoxic agents.
Asunto(s)
Sitios Frágiles del Cromosoma/efectos de los fármacos , Inestabilidad de Microsatélites/efectos de los fármacos , Mutágenos/toxicidad , Compuestos Organometálicos/toxicidad , Óxidos/toxicidad , Animales , Trióxido de Arsénico , Arsenicales , Masculino , RatasAsunto(s)
Sitios Frágiles del Cromosoma/genética , Replicación del ADN , Genoma Humano , Ácido Anhídrido Hidrolasas/genética , Afidicolina/farmacología , Transformación Celular Neoplásica/genética , Rotura Cromosómica , Sitios Frágiles del Cromosoma/efectos de los fármacos , Humanos , Linfocitos/metabolismo , Linfocitos/ultraestructura , Mitosis , Modelos Genéticos , Proteínas de Neoplasias/genética , Síndromes Neoplásicos Hereditarios/genética , Conformación de Ácido Nucleico , Estrés MecánicoRESUMEN
To gain a better understanding of the mechanism of chromosomal translocations in cancer, we investigated the spatial proximity between CBFB and MYH11 genes involved in inv(16)(p13q22) found in patients with acute myeloid leukemia. Previous studies have demonstrated a role for spatial genome organization in the formation of tumorigenic abnormalities. The nonrandom localization of chromosomes and, more specifically, of genes appears to play a role in the mechanism of chromosomal translocations. Here, two-color fluorescence in situ hybridization and confocal microscopy were used to measure the interphase distance between CBFB and MYH11 in hematopoietic stem cells (HSCs), where inv(16)(p13q22) is believed to occur, leading to leukemia development. The measured distances in HSCs were compared with mesenchymal stem cells, peripheral blood lymphocytes, and fibroblasts, as spatial genome organization is determined to be cell-type specific. Results indicate that CBFB and MYH11 are significantly closer in HSCs compared with all other cell types examined. Furthermore, the CBFB-MYH11 distance is significantly reduced compared with CBFB and a control locus in HSCs, although separation between CBFB and the control is â¼70% of that between CBFB and MYH11 on metaphase chromosomes. HSCs were also treated with fragile site-inducing chemicals because both the genes contain translocation breakpoints within these regions. However, treatment with fragile site-inducing chemicals did not significantly affect the interphase distance. Consistent with previous studies, our results suggest that gene proximity may play a role in the formation of cancer-causing rearrangements, providing insight into the mechanism of chromosomal abnormalities in human tumors.
Asunto(s)
Núcleo Celular , Subunidad beta del Factor de Unión al Sitio Principal/genética , Células Madre Hematopoyéticas/citología , Interfase/genética , Leucemia Mieloide Aguda/genética , Cadenas Pesadas de Miosina/genética , Translocación Genética , 2-Aminopurina/farmacología , Afidicolina/farmacología , Células Cultivadas , Sitios Frágiles del Cromosoma/efectos de los fármacos , Diminazeno/análogos & derivados , Diminazeno/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Leucemia Mieloide Aguda/patología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Microscopía ConfocalRESUMEN
Fragile sites are specific loci within the genome that exhibit increased tendencies for chromosome breakage. They are conserved among mammals and are also found in lower eukaryotes including yeast and fly. Many conditions, including mutations and exogenous factors, contribute to fragile site expression, but the nature of interaction among them remains elusive. Here, we investigated this by examining the combined effects of rrm3Δ, mec1 and hydroxyurea (HU), three conditions that induce fragile sites, on expression of the replication slow zone (RSZ), a type of fragile site in budding yeast. Contrary to the expectation that each factor would contribute to fragile site expression in an independent manner, we show that rrm3Δ and high concentrations of HU suppressed RSZ expression in mec1-4ts cells. Further analyses revealed that rrm3Δ suppression occurs via promotion of Sml1 degradation, whereas HU suppresses RSZ via a premature commitment to inviability. Taken together, these observations demonstrate that: (1) the yeast genome contains different types of fragile site with regard to regulation of their expression, and (2) each fragile-site-inducing condition does not act independently, but can elicit a cellular response(s) that can paradoxically prevent the expression of a specific type(s) of fragile sites.
Asunto(s)
Sitios Frágiles del Cromosoma , ADN Helicasas/metabolismo , Hidroxiurea/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sitios Frágiles del Cromosoma/efectos de los fármacos , ADN Helicasas/genética , Replicación del ADN/efectos de los fármacos , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The replication dynamics at common fragile site FRA6E has been evaluated by molecular combing and interphase fluorescent in situ hybridisation (FISH) in primary human lymphocytes cultured under normal or aphidicolin-induced stress conditions. FRA6E is one of the most frequently expressed common fragile sites of the human genome. It harbours several genes, PARK2 being regarded as the most relevant one. According to the results obtained from interphase FISH analysis, FRA6E can be considered a mid-late-replicating sequence characterised by heterogeneous replication timing. Molecular combing did not reveal specific replication parameters at the fragile site: fork rates were highly comparable to those detected at an early replicating locus (LMNB2) used as control and in very good agreement with the whole-genome data obtained in parallel. The same indication applied to the density of initiation zones, the inter-origin distances from adjacent ongoing forks, the frequencies of unidirectional forks, fork arrest events and asynchronous forks. Interestingly, PARK2 appeared embedded in an early/late replication transition zone, corresponding to intron 8 (162 kb) and to the fragility core of FRA6E. In cells exposed to aphidicolin, few forks progressing at a rather slow rate were observed, the majority of them being unidirectional, but again a specific response of the fragile site was not observed. In summary, at FRA6E the replication process is not impaired per se, but chromosome breakages occur preferentially at an early/late replication transition zone. Aphidicolin might increase the occurrence of breakage events at FRA6E by prolonging the time interval separating the replication of early and late replication domains. These results may be of general significance to address the problem of fragile site instability.
Asunto(s)
Sitios Frágiles del Cromosoma , Replicación del ADN , Afidicolina/farmacología , Ciclo Celular , Células Cultivadas , Rotura Cromosómica , Sitios Frágiles del Cromosoma/efectos de los fármacos , Fragilidad Cromosómica/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Humanos , Hibridación Fluorescente in Situ , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Common fragile sites are loci that preferentially form gaps and breaks on metaphase chromosomes when DNA synthesis is perturbed, particularly after treatment with the DNA polymerase inhibitor, aphidicolin. We and others have identified several cell cycle checkpoint and DNA repair proteins that influence common fragile site stability. However, the initial events underlying fragile site breakage remain poorly understood. We demonstrate here that aphidicolin-induced gaps and breaks at fragile sites are prevented when cells are co-treated with low concentrations of the topoisomerase I inhibitor, camptothecin. This reduction in breakage is accompanied by a reduction in aphidicolin-induced RPA foci, CHK1 and RPA2 phosphorylation, and PCNA monoubiquitination, indicative of reduced levels of single stranded DNA. Furthermore, camptothecin reduces spontaneous fragile site breakage seen in cells lacking ATR, even in the absence of aphidicolin. These data from cultured human cells demonstrate that topoisomerase I activity is required for DNA common fragile site breaks and suggest that polymerase-helicase uncoupling is a key initial event in this process.
Asunto(s)
Rotura Cromosómica/efectos de los fármacos , Sitios Frágiles del Cromosoma/efectos de los fármacos , Sitios Frágiles del Cromosoma/genética , Inhibidores Enzimáticos/farmacología , Inhibidores de Topoisomerasa I , Afidicolina/farmacología , Proteínas de la Ataxia Telangiectasia Mutada , Camptotecina/farmacología , Proteínas de Ciclo Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Replicación del ADN/efectos de los fármacos , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Triterpenos Pentacíclicos , Fosforilación/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteína de Replicación A/metabolismo , Triterpenos/farmacología , Ubiquitinación/efectos de los fármacos , Ácido BetulínicoRESUMEN
Cadmium (Cd) is a non-essential element and is a widespread environmental pollutant. Exposure to cadmium can result in cytotoxic, carcinogenic and mutagenic effects. Mutagenesis is an indicative of genetic instability and can be assayed using microsatellites instability. The aim of the present study is to investigate; based on the rat model, the effects of oral acute (single 8.8mg/kg BW, 1/10 LD(50)) and sub-chronic (2.93mg/kg BW, 1/30 LD(50), for 4 weeks) doses of cadmium chloride on microsatellite instability at D6mit3, D9mit2 and D15Mgh1 loci, which are located in three different common fragile sites (6q21, 9q32-q33 and 15p14, respectively), within rat genome. In the acute study, no microsatellite instability (MSI) was observed in all the three tested loci (D6mit3, D9mit2 and D15Mgh1). In the sub-chronic study, the MSI were observed in the three studied loci and was in the form of deletion of 2-3bp or addition of 3-6bp. These finding may indicate the sensitivity of microsatellite sequences located at the fragile sites and the sensitivity of the simple sequence repeats (SSRs) assay for the detection of small variations in DNA sequence. However, additional chronic toxicological trials are needed in order to assess genotoxic effects of chronic exposure to Cd.
Asunto(s)
Cadmio/toxicidad , Sitios Frágiles del Cromosoma/efectos de los fármacos , Inestabilidad de Microsatélites , Repeticiones de Microsatélite/efectos de los fármacos , Mutágenos/toxicidad , Animales , Ratas , Eliminación de Secuencia/efectos de los fármacosRESUMEN
Human chromosomal fragile sites are regions of the genome that are prone to DNA breakage, and are classified as common or rare, depending on their frequency in the population. Common fragile sites frequently coincide with the location of genes involved in carcinogenic chromosomal translocations, suggesting their role in cancer formation. However, there has been no direct evidence linking breakage at fragile sites to the formation of a cancer-specific translocation. Here, we studied the involvement of fragile sites in the formation of RET/PTC rearrangements, which are frequently found in papillary thyroid carcinoma (PTC). These rearrangements are commonly associated with radiation exposure; however, most of the tumors found in adults are not linked to radiation. In this study, we provide structural and biochemical evidence that the RET, CCDC6 and NCOA4 genes participating in two major types of RET/PTC rearrangements, are located in common fragile sites FRA10C and FRA10G, and undergo DNA breakage after exposure to fragile site-inducing chemicals. Moreover, exposure of human thyroid cells to these chemicals results in the formation of cancer-specific RET/PTC rearrangements. These results provide the direct evidence for the involvement of chromosomal fragile sites in the generation of cancer-specific rearrangements in human cells.
Asunto(s)
Carcinoma Papilar/genética , Sitios Frágiles del Cromosoma/genética , Roturas del ADN , Oncogenes/genética , Proteínas Proto-Oncogénicas c-ret/genética , Glándula Tiroides/patología , Neoplasias de la Tiroides/genética , Afidicolina/farmacología , Secuencia de Bases , Carcinoma Papilar/patología , Línea Celular , Sitios Frágiles del Cromosoma/efectos de los fármacos , Roturas del ADN/efectos de los fármacos , Humanos , Intrones/genética , Datos de Secuencia Molecular , Glándula Tiroides/citología , Neoplasias de la Tiroides/patología , Translocación Genética/efectos de los fármacos , Translocación Genética/genéticaRESUMEN
PURPOSE: Genetic factors are known to be important in the etiology of bipolar disorder (BD). The fragile sites (FSs) are a very interesting subject for the study of clinical disorders. The aim of this study was to evaluate fragile sites seen in patients with bipolar disorder and find a correlation between some fragile sites and bipolar disorder. PATIENTS AND METHODS: The frequencies of folate sensitive FSs were compared in short-term whole blood cultures from bipolar patients and from normal individuals. RESULTS: The rate of FS expression in the patients was considerably higher than in the controls (p < 0.001). Several chromosome regions including 1p36, 1q21, 1q32, 3p25, 7q22, 7q32, 11q23, 12q24, 13q32, 14q24, Xp22 and Xq26 were represented considerably more often in the patients than in the controls (p value between 0.001 to 0.036). Among these FSs, the sites 1p36, 1q21, 3p25, 7q22, 7q32, and 14q24 were not observed in other studies. CONCLUSION: These regions can be the most active of hot spots in the genomes of bipolar patients, and may harbor important genes associated with BD.
Asunto(s)
Trastorno Bipolar/genética , Sitios Frágiles del Cromosoma/genética , Fragilidad Cromosómica/genética , Cromosomas Humanos/genética , Adolescente , Adulto , Sitios Frágiles del Cromosoma/efectos de los fármacos , Fragilidad Cromosómica/efectos de los fármacos , Citogenética , Femenino , Ácido Fólico/farmacología , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Gene amplification is frequently found in human glioblastoma but the mechanisms driving amplifications remain to be elucidated. Hypoxia as hallmark of glioblastoma is known to be involved in the induction of fragile sites that are central to gene amplification. We analyzed the potential of hypoxia (pO2 0%) and mini hypoxia (pO2 5%) to induce fragile sites within a homogeneously staining region (HSR) at 12q14-15 in a glioblastoma cell line (TX3868). Treatment of cells by hypoxia or by mini hypoxia induced double minutes (DMs) and caused breakage of the HSR structure at 12q14-15, suggesting a novel hypoxia inducible fragile site on 12q. Treatment with aphidicolin, a known fragile site inducer, indicates that the hypoxia inducible fragile site is a common fragile site. Reintegration of amplified sequences and occurrence of anaphase-bridge-like structures shows that mini hypoxia and hypoxia are able to initiate amplification processes in human glioblastoma cells. Hypoxia as known tumor microenvironment factor is crucial for the development of amplifications in glioblastoma. The identification and characterization of novel common fragile sites induced by hypoxia will improve the understanding of mechanisms underlying amplifications in glioblastoma.
Asunto(s)
Sitios Frágiles del Cromosoma/genética , Cromosomas Humanos Par 12/genética , Amplificación de Genes/fisiología , Glioblastoma/irrigación sanguínea , Glioblastoma/genética , Animales , Afidicolina/farmacología , Secuencia de Bases , Hipoxia de la Célula/genética , Sitios Frágiles del Cromosoma/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Amplificación de Genes/efectos de los fármacos , Humanos , Ratones , Datos de Secuencia MolecularRESUMEN
The present study reports on the chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis, 2n = 50) with the aim of establishing a 'fragile site map' of the species. Totally, 400 aphidicolin-induced breakages were analyzed from eight young and clinically healthy animals, four males and four females; these breakages were localized in 106 RBG-negative chromosome bands or at the band-interband regions. The number of breakages per chromosome did not vary statistically 'among' the animals investigated but the differences among individual chromosomes were highly significant thus indicating that the chromosomal distribution of the breakages is not random and appears only partially related to chromosome length. Fragile sites were statistically determined as those chromosomal bands showing three or more breakages. In the river buffalo karyotype, 51 fragile sites were detected and localized on the standardized ideogram of the species. The most fragile bands were as follows: 9q213 with 24 breakages out of 400; 19q21 with 16, 17q21 and inacXq24 with 15, 15q23 with 13 and 13q23 with 12 breaks, respectively. Previous gene mapping analysis in this species has revealed that the closest loci to these fragile sites contain genes such as RASA1 and CAST (9q214), NPR3 and C9 (19q19), PLP and BTK (Xq24-q25), OarCP09 (15q24), and EDNRB (13q22) whose mutations are responsible for severe phenotypic malformations and immunodeficiency in humans as well as in mice and meat quality in pigs. Further cytogenetic and molecular studies are needed to fully exploit the biological significance of the fragile sites in karyotype evolution of domestic animals and their relationships with productive and reproductive efficiency of livestock.
Asunto(s)
Afidicolina/farmacología , Búfalos/genética , Sitios Frágiles del Cromosoma/efectos de los fármacos , Sitios Frágiles del Cromosoma/genética , Animales , Células Cultivadas , Bandeo Cromosómico/veterinaria , Rotura Cromosómica/efectos de los fármacos , Mapeo Cromosómico/veterinaria , Femenino , Cariotipificación/veterinaria , Masculino , Cromosoma X/efectos de los fármacos , Cromosoma X/genéticaRESUMEN
Fragile sites are heritable specific chromosome loci that exhibit an increased frequency of gaps, poor staining, constrictions or breaks when chromosomes are exposed to partial DNA replication inhibition. They constitute areas of chromatin that fail to compact during mitosis. They are classified as rare or common depending on their frequency within the population and are further subdivided on the basis of their specific induction chemistry into different groups differentiated as folate sensitive or non-folate sensitive rare fragile sites, and as aphidicolin, bromodeoxyuridine (BrdU) or 5-azacytidine inducible common fragile sites. Most of the known inducers of fragility share in common their potentiality to inhibit the elongation of DNA replication, particularly at fragile site loci. Seven folate sensitive (FRA10A, FRA11B, FRA12A, FRA16A, FRAXA, FRAXE and FRAXF) and two non-folate sensitive (FRA10B and FRA16B) fragile sites have been molecularly characterized. All have been found to represent expanded DNA repeat sequences resulting from a dynamic mutation involving the normally occurring polymorphic CCG/CGG trinucleotide repeats at the folate sensitive and AT-rich minisatellite repeats at the non-folate sensitive fragile sites. These expanded repeats were demonstrated, first, to have the potential, under certain conditions, to form stable secondary non-B DNA structures (intra-strand hairpins, slipped strand DNA or tetrahelical structures) and to present highly flexible repeat sequences, both conditions which are expected to affect the replication dynamics, and second, to decrease the efficiency of nucleosome assembly, resulting in decondensation defects seen as fragile sites. Thirteen aphidicolin inducible common fragile sites (FRA2G, FRA3B, FRA4F, FRA6E, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA8C, FRA9E, FRA16D and FRAXB) have been characterized at a molecular level and found to represent relatively AT-rich DNA areas, but without any expanded repeat motifs. Analysis of structural characteristics of the DNA at some of these sites (FRA2G, FRA3B, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA16D and FRAXB) showed that they contained more areas of high DNA torsional flexibility with more highly AT-dinucleotide-rich islands than neighbouring non-fragile regions. These islands were shown to have the potential to form secondary non-B DNA structures and to interfere with higher-order chromatin folding. Therefore, a common fragility mechanism, characterized by high flexibility and the potential to form secondary structures and interfere with nucleosome assembly, is shared by all the cloned classes of fragile sites. From the clinical point of view, the folate sensitive rare fragile site FRAXA is the most important fragile site as it is associated with the fragile X syndrome, the most common form of familial mental retardation, affecting about 1/4000 males and 1/6000 females. Mental retardation in this syndrome is considered as resulting from the abolition of the FMR1 gene expression due to hypermethylation of the gene CpG islands adjacent to the expanded methylated trinucleotide repeat. FRAXE is associated with X-linked non-specific mental retardation, and FRA11B with Jacobsen syndrome. There is also some evidence that fragile sites, especially common fragile sites, are consistently involved in the in vivo chromosomal rearrangements related to cancer, whereas the possible implication of common fragile sites in neuropsychiatric and developmental disorders is still poorly documented.
Asunto(s)
Sitios Frágiles del Cromosoma , Fragilidad Cromosómica , Secuencia de Bases , Sitios Frágiles del Cromosoma/efectos de los fármacos , Fragilidad Cromosómica/efectos de los fármacos , Citogenética , ADN/genética , Distamicinas/farmacología , Femenino , Ácido Fólico/farmacología , Síndrome del Cromosoma X Frágil/genética , Expresión Génica , Humanos , Masculino , Expansión de Repetición de TrinucleótidoRESUMEN
An investigation to understand the dynamics and biological significance of fragile site expression, and identification of 5-fluorodeoxyuridine (FUdR) induced chromosomal gaps/breaks, were carried out in an experimental flock of 45 Suffolk sheep. The statistical comparison revealed, highly significant variation in the frequency of chromosomal fragile site expression between control and FUdR cultures. Mean (+/- S.D.) values for cells with gaps and breaks, or aberrant cell count (AC), and the number of aberrations (NoA) per animal were 2.02 +/- 0.34, 2.42 +/- 0.48, 13.26 +/- 0.85 and 21.87 +/- 1.88 (P lessthan 0.01) in control and FUdR cultures, respectively. The comparison of age revealed nonsignificant variation between control and FUdR cultures. The G-band analysis of fragile site data revealed gaps in 29 autosomal and two X-chromosomal bands in the control cultures, whereas FUdR treated cultures scored 78 unstable bands in autosomes of which 56 were significantly fragile. X-chromosomes expressed breaks and gaps in six G-negative bands and five of them (Xq13, Xq15, Xq17, Xq24 and Xq26) were significantly fragile. The distribution comparison of autosomal fragile sites between sex groups did not reveal any significant variation. Female X-chromosomes were significantly more fragile than the male X-chromosomes. The distribution comparison for age groups (lambs versus adults) revealed significantly higher number of fragile bands in adults. Comparison of published data on reciprocal translocations in sheep with the fragile-site data obtained in this study indicated that the break sites of both phenomena were correlated. Similarities were also found between fragile sites and breakpoints of evolutionary significance in family Bovidae.
Asunto(s)
Sitios Frágiles del Cromosoma/efectos de los fármacos , Floxuridina/farmacología , Ácido Fólico/farmacología , Oveja Doméstica/genética , Animales , Recuento de Células , Aberraciones Cromosómicas/efectos de los fármacos , Bandeo Cromosómico , Cromosomas de los Mamíferos/genética , Secuencia Conservada , Cruzamientos Genéticos , Evolución Molecular , Femenino , Genoma/genética , Cariotipificación , Masculino , Translocación Genética/efectos de los fármacos , Reino Unido , Cromosoma X/genéticaRESUMEN
We report on the molecular basis of the rare, folate-sensitive fragile site FRA11A in chromosome band 11q13 in a family with cytogenetic expression. Five individuals express the fragile site and one was mentally retarded. Expansion of a polymorphic CGG-repeat located at the 5' end of the C11orf80 gene causes FRA11A. The CGG-repeat elongation coincides with hypermethylation of the adjacent CpG island and subsequent transcriptional silencing of the C11orf80 gene. This gene has no homology with known genes. A relationship between cytogenetic expression of the fragile site and the mental handicap seems unlikely, as FRA11A was found in a mentally retarded patient as well as in phenotypically normal carriers from the same family. However, incomplete penetrance cannot be entirely excluded.
Asunto(s)
Sitios Frágiles del Cromosoma/efectos de los fármacos , Sitios Frágiles del Cromosoma/genética , Cromosomas Humanos Par 11/genética , Ácido Fólico/farmacología , Biología Computacional , Femenino , Regulación de la Expresión Génica/genética , Humanos , Hibridación Fluorescente in Situ , Masculino , Linaje , FenotipoRESUMEN
Fragile sites are specific genomic loci that form gaps, constrictions and breaks on chromosomes exposed to replication stress conditions. In the father of a patient with Beckwith-Wiedemann syndrome and a pure truncation of 18q22-qter, a new aphidicolin-sensitive fragile site on chromosome 18q22.2 (FRA18C) is described. The region in 18q22 appears highly enriched in flexibility islands previously found to be the characteristic of common fragile site regions. The breakpoint was cloned in this patient. The break disrupts the DOK6 gene and was immediately followed by a repetitive telomere motif, (TTAGGG)(n). Using fluorescent in situ hybridisation, the breakpoint in the daughter was found to coincide with the fragile site in the father. The breakpoint region was highly enriched in AT-rich sequences. It is the first report of an aphidicolin-sensitive fragile site that coincides with an in vivo chromosome truncation in the progeny.
Asunto(s)
Afidicolina/farmacología , Rotura Cromosómica/efectos de los fármacos , Sitios Frágiles del Cromosoma/efectos de los fármacos , Sitios Frágiles del Cromosoma/genética , Cromosomas Humanos Par 18/efectos de los fármacos , Cromosomas Humanos Par 18/genética , Secuencia de Bases , Niño , Deleción Cromosómica , Clonación Molecular , Análisis Mutacional de ADN , Padre , Femenino , Humanos , Datos de Secuencia Molecular , Linaje , Secuencias Repetitivas de Ácidos Nucleicos/genéticaRESUMEN
OBJECTIVE: To report fragility at 10q23.3 in a fetus exposed to phenytoin during pregnancy. Review of the literature. METHODS: Amniocytes were cultured in A10 (WISENT) culture medium. Molecular polymorphism studies of MTHFR gene using PCR were performed on fetal tissues. RESULTS: The fragile site was expressed in all 22 amniocyte colonies analyzed. Analysis of fetal blood showed 46,XX[98]/46,XX,fra(10)(q23.3)[3]/46,XX,del(10)(q23.3) [1]. Molecular studies of the MTHFR (methylenetetrahydrofolate reductase) gene identified a compound heterozygote genotype for two polymorphisms, 677C>T and 1298A>C. CONCLUSION: The fragility at 10q23.3 is unlikely to be due to culture condition-induced folic acid deficiency (medium contains folate). It is possible that this finding represents a previously undescribed folic acid-insensitive fragile site in the region of 10q23.3. Alternatively, the fetal cells may have had decreased folate metabolism, and the fragile site was the known folate-sensitive FRA10A. Since phenytoin has been shown to decrease MTHFR activity in mice, we postulate that the fragile site at 10q23.3 in this fetus may have arisen secondary to a combination of the polymorphisms in MTHFR and exposure to this drug, and is indeed FRA10A.