RESUMEN
The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.
Asunto(s)
Gangliosidosis GM2 , Enfermedad de Tay-Sachs , Desoxirribonucleasa I/metabolismo , Fibroblastos/metabolismo , Proteína Activadora de G (M2) , Gangliósido G(M2)/genética , Gangliósido G(M2)/metabolismo , Gangliosidosis GM2/genética , Gangliosidosis GM2/metabolismo , Gangliosidosis GM2/terapia , Edición Génica , Globósidos/metabolismo , Glicosaminoglicanos/metabolismo , Hexosaminidasa A/metabolismo , Humanos , Lipopolisacáridos/metabolismo , Liposomas/metabolismo , Enfermedad de Tay-Sachs/genética , Enfermedad de Tay-Sachs/metabolismo , Enfermedad de Tay-Sachs/terapia , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Chromatin accessibility is directly linked with transcription in eukaryotes. Accessible regions associated with regulatory proteins are highly sensitive to DNase I digestion and are termed DNase I hypersensitive sites (DHSs). DHSs can be identified by DNase I digestion, followed by high-throughput DNA sequencing (DNase-seq). The single-base-pair resolution digestion patterns from DNase-seq allows identifying transcription factor (TF) footprints of local DNA protection that predict TF-DNA binding. The identification of differential footprinting between two conditions allows mapping relevant TF regulatory interactions. Here, we provide step-by-step instructions to build gene regulatory networks from DNase-seq data. Our pipeline includes steps for DHSs calling, identification of differential TF footprints between treatment and control conditions, and construction of gene regulatory networks. Even though the data we used in this example was obtained from Arabidopsis thaliana, the workflow developed in this guide can be adapted to work with DNase-seq data from any organism with a sequenced genome.
Asunto(s)
Cromatina/metabolismo , Mapeo Cromosómico/métodos , Huella de ADN/métodos , Desoxirribonucleasa I/metabolismo , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Arabidopsis/genética , Arabidopsis/metabolismo , Cromatina/genética , Genómica , Unión Proteica , Programas Informáticos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
During the interphase the nuclear DNA of metazoan cells is organized in supercoiled loops anchored to constituents of a nuclear substructure or compartment known as the nuclear matrix. The stable interactions between DNA and the nuclear matrix (NM) correspond to a set of topological relationships that define a nuclear higher-order structure (NHOS). Current evidence suggests that the NHOS is cell-type-specific. Biophysical evidence and theoretical models suggest that thermodynamic and structural constraints drive the actualization of DNA-NM interactions. However, if the topological relationships between DNA and the NM were the subject of any biological constraint with functional significance then they must be adaptive and thus be positively selected by natural selection and they should be reasonably conserved, at least within closely related species. We carried out a coarse-grained, comparative evaluation of the DNA-NM topological relationships in primary hepatocytes from two closely related mammals: rat and mouse, by determining the relative position to the NM of a limited set of target sequences corresponding to highly-conserved genomic regions that also represent a sample of distinct chromosome territories within the interphase nucleus. Our results indicate that the pattern of topological relationships between DNA and the NM is not conserved between the hepatocytes of the two closely related species, suggesting that the NHOS, like the karyotype, is species-specific.
Asunto(s)
ADN/ultraestructura , Hepatocitos/citología , Matriz Nuclear/ultraestructura , Animales , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Hepatocitos/fisiología , Cinética , Masculino , Ratones Endogámicos , Matriz Nuclear/genética , Ratas Wistar , Especificidad de la EspecieRESUMEN
Many mechanisms involved in sperm-mediated gene transfer (SMGT) are still unknown. It is still a matter of debate whether exogenous DNA fragments incorporated by the embryo are originated from those bound to the sperm membrane or by those that penetrated the intracellular compartment. In an attempt to elucidate the transmission mechanism of exogenous DNA molecules by sperm, some authors suggested a treatment with DNAse I to remove DNA molecules outside the sperm. But little is known regarding the effects of DNAse I treatment on sperm viability and its impact on sperm organelles. An important aspect of the SMGT technique is the amount of exogenous DNA incubated with sperm, which may influence the internalization rate. Due to the inconsistencies found in literature, this work aimed to contribute to bovine sperm physiology knowledge evaluating the effects of different DNA concentrations, electroporation, and DNAse I treatments on sperm viability characteristics, DNA uptake, and IVF. For that, the effects of different concentrations of exogenous DNA (250, 500 and 1000 ng/10(6) cells) and incubation or electroporation were tested on sperm functional characteristics and in vitro embryo production. No effect of DNA concentration was observed on uptake, plasma membrane integrity, and mitochondrial membrane potential. The addition of exogenous DNA induced a decrease on acrosomal lesion in the 500-ng group when compared to the control. Cells incubated with DNA, electroporated, and treated with DNAse I presented a deleterious influence on mitochondrial membrane potential. In vitro fertilization was made with 1000 ng of DNA, sperm cells incubated or electroporated followed by DNAse I treatment. No significant difference was found in cleavage rate. Blastocyst rates were 24.36% for the control; 19.65% for incubated; 3.5% for electroporated control; and 17.40% for electroporated. There is a significant difference in blastocyst rate between the control and electroporated control groups. The incubated group yielded five and electroporated two positive blastocysts evaluated by epifluorescence microscopy. Polymerase chain reaction screening shows 17% of positive embryos for incubation and 11% for electroporation. Fluorescence in situ hybridization showed the presence of exogenous gene in embryos. These results show that exogenous DNA molecules can be conducted by an intracellular mechanism. The SMGT protocol using electroporation and DNAse I treatment reduces sperm mitochondrial function, in vitro embryo production and increases sperm DNA fragmentation.
Asunto(s)
Bovinos/fisiología , Desoxirribonucleasa I/metabolismo , Electroporación/veterinaria , Espermatozoides/fisiología , Animales , Supervivencia Celular , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Masculino , PlásmidosRESUMEN
Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the causative agent of cholera. Recently, it was found that V. cholerae isolates from the Haiti outbreak were poorly transformed by this mechanism. Here, we show that an integrating conjugative element (ICE)-encoded DNase, which we name IdeA, is necessary and sufficient for inhibiting natural transformation of Haiti outbreak strains. We demonstrate that IdeA inhibits this mechanism of HGT in cis via DNA endonuclease activity that is localized to the periplasm. Furthermore, we show that natural transformation between cholera strains in a relevant environmental context is inhibited by IdeA. The ICE encoding IdeA is globally distributed. Therefore, we analyzed the prevalence and role for this ICE in limiting natural transformation of isolates from Bangladesh collected between 2001 and 2011. We found that IdeA(+) ICEs were nearly ubiquitous in isolates from 2001 to 2005; however, their prevalence decreased to â¼40% from 2006 to 2011. Thus, IdeA(+) ICEs may have limited the role of natural transformation in V. cholerae. However, the rise in prevalence of strains lacking IdeA may now increase the role of this conserved mechanism of HGT in the evolution of this pathogen.
Asunto(s)
Proteínas Bacterianas/genética , Transferencia de Gen Horizontal , Secuencias Repetitivas Esparcidas , Transformación Bacteriana , Vibrio cholerae/genética , Proteínas Bacterianas/fisiología , Bangladesh , Quitina/química , Cólera/genética , Cólera/microbiología , Conjugación Genética , ADN/metabolismo , ADN Bacteriano/genética , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasas/química , Evolución Molecular , Haití , Humanos , Operón Lac , Modelos Genéticos , Mutación , Periplasma/metabolismo , Filogenia , Prevalencia , Vibrio cholerae/metabolismo , beta-Lactamasas/metabolismoRESUMEN
OBJECTIVES: DNA methylation plays a critical role in the regulation of the transcription of the suppressors of cytokine signaling (SOCS) 1 and SOCS3, which are modulators in the inflammation. We hypothesized that the methylation status of SOCS1, SOCS3, and long interspersed nuclear element (LINE)-1 in gingival tissues previously inflamed would be similar to that found in gingival tissues without clinical inflammation in the period studied. MATERIALS AND METHODS: Laser capture microdissection was performed to isolate epithelial and connective gingival tissues. The groups were comprised by ten patients without history of periodontitis and absence of clinical signs of inflammation in the gingiva during the study (healthy group) and ten patients with history of periodontitis, presenting inflammation in the gingival tissue at the first examination of the study (controlled chronic periodontitis group). The gingival biopsies from the controlled chronic periodontitis group were collected after controlling the inflammation. DNA methylation patterns were analyzed using methylation-specific high-resolution melting and combined bisulfite restriction analysis. RESULTS: DNA methylation levels for SOCS1 and SOCS3 did not differ between groups or tissues; likewise, no differences were observed in total LINE-1 methylation or at specific loci. CONCLUSION: At 3 months following control of inflammation in gingival tissues, the methylation profile of SOCS1, SOCS3, and LINE-1 is similar between connective and epithelial tissues from patients that were previously affected or not by chronic inflammation. CLINICAL RELEVANCE: Clinical results of a successful treatment are observed after inflammation control and the molecular findings illustrate local and general methylation patterns in recovering tissues toward health conditions and might help to understand events that are occurring in oral cells.
Asunto(s)
Metilación de ADN , Desoxirribonucleasa I/metabolismo , Encía/metabolismo , Periodontitis/metabolismo , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Biopsia , Brasil , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Human adenoviruses (HAdVs) are the second-leading cause of childhood gastroenteritis worldwide. This virus is commonly found in environmental waters and is very resistant to water disinfection and environmental stressors, especially UV light inactivation. Molecular techniques, such as PCR-based methods (Polymerase Chain Reaction), are commonly used to detect and identify viral contamination in water, although PCR alone does not allow the discrimination between infectious and non-infectious viral particles. A combination of cell culture and PCR has allowed detection of infectious viruses that grow slowly or fail to produce cytopathic effects (CPE) in cell culture. This study aimed to assess the integrity and viability of human adenovirus (HAdV) in environmental water and evaluate circulating strains by molecular characterization in three sites of the water supply in Florianópolis, Santa Catarina Island, Brazil: Peri Lagoon water, spring source water, and water from the public water supply system. METHODS: Water samples were collected, concentrated and HAdV quantified by real-time PCR. Viral integrity was evaluated by enzymatic assay (DNase I) and infectivity by plaque assay (PA) and integrated cell culture using transcribed mRNA (ICC-RT-qPCR). Samples containing particles of infectious HAdV were selected for sequencing and molecular characterization. RESULTS: The analyzed sites contained 83, 66 and 58% undamaged HAdV particles (defined as those in which the genetic material is protected by the viral capsid) at Peri Lagoon, spring source water and public supply system water, respectively. Of these, 66% of the particles (by PA) and 75% (by ICC-RT-qPCR) HAdV were shown to be infectious, due to being undamaged in Peri Lagoon, 33% (by PA) and 58% (by ICC-RT-qPCR) in spring source water and 8% (by PA) and 25% (by ICC-RT-qPCR) in the public water supply system. ICC-RT-qPCR, a very sensitive and rapid technique, was able to detect as low as 1 × 102 HAdV genome copies per milliliter of infectious viral particles in the environmental water samples. The molecular characterization studies indicated that HAdV-2 was the prevalent serotype. CONCLUSIONS: These results indicate a lack of proper public health measures. We suggest that HAdV can be efficiently used as a marker of environmental and drinking water contamination and ICC-RT-qPCR demonstrated greater sensitivity and speed of detection of infectious viral particles compared to PA.
Asunto(s)
Adenovirus Humanos/aislamiento & purificación , Adenovirus Humanos/fisiología , Agua Potable/virología , Viabilidad Microbiana , Adenovirus Humanos/genética , Brasil , ADN Viral/metabolismo , Desoxirribonucleasa I/metabolismo , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Carga ViralRESUMEN
Beauveria bassiana strains from different hosts and geographic origins were assayed for the presence of double-stranded RNA (dsRNA). Two of them (15.4%) showed extra bands, with approximately 4.0-3.5 kb and 2-0.7 kb, respectively, after electrophoretic separation of undigested nucleic acids. Virus-like particles were approximately 28-30 nm diam. The dsRNA was maintained after conidiogenesis (vertical transmission) and was transmitted horizontally by hyphal anastomosis. Strains purged of dsRNA obtained after cycloheximide treatment showed increased conidial production when compared with strains carrying dsRNA particles. Bioassays demonstrated hypovirulence associated with dsRNA. The mean mortality against the insect Euschistus heros was reduced in strains containing dsRNA when compared with the isogenic dsRNA-free ones.
Asunto(s)
Beauveria/patogenicidad , Beauveria/virología , Infecciones por Virus ARN/virología , Virus ARN/aislamiento & purificación , ARN Bicatenario/aislamiento & purificación , Animales , Beauveria/ultraestructura , Desoxirribonucleasa I/metabolismo , Insectos , Microscopía Electrónica de Transmisión , Virus ARN/genética , Virus ARN/metabolismo , Virus ARN/ultraestructura , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/ultraestructura , ARN Viral/química , ARN Viral/genética , Técnica del ADN Polimorfo Amplificado Aleatorio , Ribonucleasa Pancreática/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , VirulenciaRESUMEN
PURPOSE: Nuclear loss is a most remarkable organelle disappearance during terminal differentiation of lens fiber cells given that it implicates the full degradation of a major molecular component, DNA. Consequently, to gain insight into the progression of DNA cleavage we analyzed the appearance of single strand breaks in relationship with chromatin condensation. To assess a possible involvement of DNase I in DNA fragmentation we explored its localization in lens fibers having different degrees of nuclear breakdown, evaluated by the state of chromatin, nuclear envelope, and DNA. METHODS: Whole mounts of adult bovine lens epithelium as well as lens cryosections were utilized to examine, using antibodies or specific molecular probes, the localization of DNase I, nuclear membrane, lamins, and DNA 3'-OH-free termini. Nuclease activity gel and western blot assays were used to characterize DNase I in different lens fiber extracts. RESULTS: Nuclear morphology was found to undergo significant changes from the onset of fiber differentiation. Initial spherical nuclei present at early fibergenesis stages evolve to elongated ones in mature fibers. Chromatin did not present signs of condensation in these nuclei. However, nuclei from fibers located deeper in lens volume exhibited some chromatin condensation and fragmentation while the nuclear lamina appeared undamaged. At more advanced stages, different patterns of nuclear envelope integrity and chromatin condensation and cleavage were observed. DNase I was found in the cytoplasm in the very initial fibers and then in the nuclear territory. DNase I appeared closely associated with fully condensed and fragmented chromatin at the final phases of nuclear breakdown. CONCLUSIONS: DNase I is a nuclease present in bovine lens fibers and can be considered as an enzyme producing final DNA cleavage since it is closely associated with highly fragmented DNA in disintegrating nuclei.
Asunto(s)
Diferenciación Celular/fisiología , Cromatina/enzimología , Fragmentación del ADN , Desoxirribonucleasa I/metabolismo , Cristalino/citología , Cristalino/enzimología , Animales , Western Blotting , Bovinos , Núcleo Celular/enzimología , Electroforesis en Gel de Poliacrilamida , Células Epiteliales/enzimología , Técnica del Anticuerpo Fluorescente Indirecta , Etiquetado Corte-Fin in Situ , Membrana Nuclear/enzimologíaRESUMEN
Genomic DNA sequencing and alignment with the known DNase I mRNA showed that the bovine gene consists of 9 exons and that only the last 8 encode the protein, since initial ATG was found at exon II. RT-PCR was used to identify DNase I mRNA in lens epithelium in vivo and in cultured epithelial cells. We found DNase I transcripts having the same nucleotide sequence as the pancreas form and others lacking almost all exon V. The lens protein presented a slightly higher relative molecular weight than the pancreatic enzyme. Lens DNase I was located in secretory pathway organelles and excluded from the nucleus. Nevertheless, in apoptotic lens epithelial cells in vitro, DNase I translocated to the nucleus and co-localized with TUNEL positive chromatin aggregates. These results indicate that cells in the lens epithelium constitutively express DNase I, and suggest a direct involvement of this nuclease in the final phases of chromatin degradation.
Asunto(s)
Apoptosis/fisiología , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Células Epiteliales/enzimología , Regulación Enzimológica de la Expresión Génica/fisiología , Cristalino/enzimología , ARN Mensajero/metabolismo , Animales , Apoptosis/efectos de los fármacos , Secuencia de Bases , Bovinos , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Cristalino/efectos de los fármacos , Datos de Secuencia Molecular , ARN Mensajero/genética , Estaurosporina , Distribución Tisular/efectos de los fármacosRESUMEN
Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.
Asunto(s)
Piscirickettsiaceae/aislamiento & purificación , Salmón/microbiología , Animales , Técnicas Bacteriológicas , Línea Celular , Centrifugación por Gradiente de Densidad , ADN Bacteriano/análisis , ADN Bacteriano/aislamiento & purificación , Desoxirribonucleasa I/metabolismo , Enfermedades de los Peces/microbiología , Microscopía Fluorescente , Piscirickettsiaceae/genética , Infecciones por Piscirickettsiaceae/microbiología , Infecciones por Piscirickettsiaceae/veterinaria , Reacción en Cadena de la Polimerasa , Ácidos TriyodobenzoicosRESUMEN
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
BACKGROUND AND OBJECTIVES: Analysis of DNA polymorphic sites is a powerful tool for detection of gene flow in human evolutionary studies and to trace genetic background associated with abnormal genes. The beta-globin locus contains more than 20 single-base restriction fragment length polymorphism (RFLP) sites spanning over 80 kb on chromosome 11. Far downstream of the expressed genes, there is a hypersensitive site (HS). The function of the 3'-HS remains unknown. As an approach to the understanding of the 3'-HS region in sickle cell anemia we searched for sequence polymorphism in the AT-rich region, using a non-radioactive polymerase chain reaction (PCR)-single strand conformational polymorphism (SSCP) technique. DESIGN AND METHODS: A 460 bp fragment located at the 3' of the b globin gene was amplified from patients (with sickle cell anemia and HbSC disease), and from AS individuals. Standard RFLP-haplotyping was performed and compared with the PCR-SSCP screening strategy. RESULTS: Two distinct band patterns were revealed by SSCP testing, each one in strict linkage disequilibrium with either Benin or Bantu haplotypes. Direct sequencing of the amplified segment revealed a TAA insertion in the AT-rich region, in all 121 beta(S) Benin chromosomes tested, but not in other beta(S) haplotypes from the total of 380 beta(S) chromosomes typed. INTERPRETATION AND CONCLUSIONS: SSCP analysis could easily distinguish sequence variations in the 3'AT-rich region of the beta-globin cluster, and a TAA insertion in this region seems to be specific for the Benin-beta(S) chromosome.
Asunto(s)
Anemia de Células Falciformes/genética , Globinas/genética , Mutagénesis Insercional/genética , Secuencia Rica en At/genética , Secuencia de Bases , Sitios de Unión , Desoxirribonucleasa I/metabolismo , Variación Genética , Humanos , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Trypanosoma cruzi is the etiological agent of Chagas. Although the nuclear chromatin of this parasite is organized in the form of nucleosome filaments, its chromatin is physically and enzymatically fragile, and no condensation into chromosomes occurs during mitosis. All previous investigations have been carried out with epimastigote form in its proliferate stage. It is not known whether these differences in chromatin structure are also found in the non-proliferate stationary epimastigote forms and in tissue derived trypomastigotes. Our results confirm that chromatin of logarithmic epimastigotes presents limited compaction when increasing salt concentrations from 1 to 100 mM NaCl, and no 30-nm fibers were formed. Contrary to these results, non-proliferative forms of the parasites showed a pattern of compactation similar to that observed in rat liver chromatin, where solenoids of 30-nm fibers are formed at 100-mM NaCl. In accordance with these results, digestion of the nuclear chromatin with DNase I revealed that the chromatin of logarithmic phase epimastigotes was more accessible to the enzyme. We conclude from these results that structural differences in the chromatin exist not only between T. cruzi and higher eukaryotes but also among various forms of the parasite. The functional significance of these differences are currently under investigation.
Asunto(s)
Núcleo Celular/ultraestructura , Cromatina/ultraestructura , Estadios del Ciclo de Vida/fisiología , Nucleosomas/ultraestructura , Trypanosoma cruzi/citología , Animales , Desoxirribonucleasa I/metabolismo , Cloruro de Sodio/farmacología , Trypanosoma cruzi/genéticaRESUMEN
The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA-protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.
Asunto(s)
Glicoproteínas/metabolismo , Proteínas Gestacionales/genética , Glicoproteínas beta 1 Específicas del Embarazo , Transcripción Genética , Animales , Células COS , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/metabolismo , Huella de ADN , Desoxirribonucleasa I/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Glicoproteínas/genética , Células HeLa , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Familia de Multigenes , Placenta/metabolismo , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Timidina Quinasa/genética , TransfecciónRESUMEN
Chromosome breakpoints induced by neutrons or gamma rays in Chinese hamster ovary cells were mapped to Giemsa-light or Giemsa-dark bands or to band junctions. Radiation-induced breakpoints were found to be distributed nonrandomly according to chromosome or band length. More than 60% of the breakpoints were localized in G-light bands. A group of 13 bands which corresponded to only 7% of the total chromosome length contained 22% of the breakpoints produced by neutrons and 14% of those induced by gamma rays. Seven of these 13 bands are also preferentially damaged by AluI, BamHI and DNase I as reported previously. The results indicate that chromatin and nuclear structure may play a role in the distribution of breakpoints produced by ionizing radiation and endonucleases.
Asunto(s)
Aberraciones Cromosómicas , Rayos gamma , Neutrones , Animales , Células CHO , Bandeo Cromosómico , Cricetinae , Cricetulus , Desoxirribonucleasa BamHI/metabolismo , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Mapeo Restrictivo , Fase SRESUMEN
The biosynthesis of met-enkephalin in human pituitary and human pituitary adenomas is still not well known. In this work, we studied the processing of proenkephalin-derived peptides in postmortem human pituitary (PMHP), ACTH-producing adenomas (ACTH-PA), nonfunctioning adenomas (NFA), and GH-producing adenomas (GH-PA). ACTH-PA contained at least 10 times more proenkephalin-derived peptides than PMHP, NFA,and GH-PA. Proenkephalin processing was different in the four tested tissues. In ACTH-PA, proenkephalin was processed to high-, intermediate-, and low-mol-wt products. The highest met-enkephalin-containing peptides levels corresponded to intermediate and low-mol-wt materials, although met-enkephalinArg-Phe and synenkephalin immunoreactivity appeared only in high-mol-wt peptides. In PMHP and NFA, met-enkephalin-Arg-Phe immunoreactivity was detected in intermediate- and low-mol-wt materials, and it was absent in GH-PA. Immunoblotting of ACTH-PA showed that met-enkephalin-Arg-Phe immunoreactivity corresponded to peptides of 44, 32-30, 27, and 17 kDa. The 32-30 and 17-kDa molecules were localized in the nuclear fraction where they were extracted after enzymatic digestion with DNase I. Plasmatic met-enkephalin levels did not increase in patients with Cushing's disease, suggesting that the pentapeptide stored in ACTH-PA was not released to the general circulation. In conclusion, we demonstrated that only ACTH-PA contained high levels of proenkephalin peptides, which were stored in cytoplasm organelles and in the nucleus, probably bound to chromatin. These results suggest an adenoma-specific physiological role of proenkephalin products.
Asunto(s)
Adenoma/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Encefalinas/metabolismo , Neoplasias Hipofisarias/metabolismo , Precursores de Proteínas/metabolismo , Adulto , Anciano , Carboxipeptidasa B , Carboxipeptidasas/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Desoxirribonucleasa I/metabolismo , Encefalina Metionina/metabolismo , Femenino , Hormona de Crecimiento Humana/metabolismo , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Peso Molecular , Hipófisis/metabolismo , Procesamiento Proteico-Postraduccional , Tripsina/metabolismoRESUMEN
DNase I was electroporated into S-phase CHO cells and induced chromosome breakpoints were localized in G-banded metaphases. More than 75% of breakpoints mapped to Giemsa-light bands, 18% to Giemsa-dark bands and about 7% to band junctions. Chromosome breakpoint clusters produced by DNase I colocalized with chromosome breakpoints induced by the restriction endonucleases AluI and BamHI in the G1- and S-phases of the cell cycle in CHO cells. Digestion of metaphase spreads with AluI, BamHI and DNase I produced G-bands, indicating that G-light bands are more sensitive to endonuclease action. The possible role of nuclease-sensitive sites in active chromatin as selective targets for the induction of chromosome breakpoints by these endonucleases is discussed.
Asunto(s)
Rotura Cromosómica , Desoxirribonucleasa I/metabolismo , Animales , Células CHO , Bandeo Cromosómico , Cromosomas/metabolismo , Cricetinae , Desoxirribonucleasa BamHI/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Electroporación , Fase S/genéticaRESUMEN
Actin has been described in all eukaryotic cells as the major microfilament cytoskeletal protein. Although prokaryotic cells do not have a cytoskeleton, proteins related to the latter have been found in different prokaryotic species. We have found prokaryotic actin-related proteins in the enterobacterium Escherichia coli and in the cyanobacteria Anabaena cylindrica and Anabaena variabilis. They were identified by the following criteria: (1) by cross-reaction with a fluorescent conjugated anti-actin (rat-brain) mAb by Western blot analysis (in total cellular extracts); (2) specific binding of acetone powder and soluble cellular extracts to DNase I; and (3) specific binding of cells and total cellular extracts to phalloidin. In E coli, specific binding of phalloidin labelled with rhodamine to cells was detected by spectrofluorometry. In total cellular extracts, three bands of 60, 43 and 35 kDa were weakly recognized by the mAb by Western blot analysis; this recognition increased when phalloidin was added to the extracts. Furthermore, three polypeptides of kDa were isolated by binding to DNase I, showing pI values of 6.7, 6.65 and 6.6, less acidic than all reported actin pI values. In A. cylindrica and A. variabilis, specific binding of phalloidin labelled with rhodamine to cells was also detected by spectrofluorometry. In total and soluble cellular extracts, the mAb recognized two bands of 45 and 40 kDa by Western blot analysis, but only the first was purified by binding to DNase I, and it showed three isoforms of pI values 6.8, 6.5 and 6.4. These results suggest the presence, in prokaryotes, of proteins with similar biochemical characteristics to eukaryotic actin.
Asunto(s)
Actinas/química , Anabaena/química , Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/química , Proteínas de Plantas/aislamiento & purificación , Actinas/inmunología , Anabaena/inmunología , Anabaena/ultraestructura , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Biopolímeros , Western Blotting , Reacciones Cruzadas , Desoxirribonucleasa I/metabolismo , Escherichia coli/inmunología , Escherichia coli/ultraestructura , Evolución Molecular , Faloidina/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Unión Proteica , Ratas , Especificidad de la EspecieRESUMEN
In the fibronectin gene promoter the cAMP response element (CRE) and the CCAAT box are separated by only 20 base pairs (bp), i.e. two turns of double helix. Binding of nuclear proteins to these elements, assessed by DNase I footprinting, differs in the different cell types. While in a variety of cells tested (HeLa, granulosa, brain, and adenocarcinoma) only CRE binding activity is observed, liver extracts show both CRE and CCAAT binding activities. Competitions with CRE oligonucleotides were able to prevent the binding of both liver factors, while competitions with CCAAT oligonucleotides only abolished the binding to the CCAAT box. Consistently, the occupation of the CCAAT box was reduced when the distance between the CRE and CCAAT elements was increased in a series of spacing mutants in which DNA fragments of 20, 28, or 44 bp were inserted, and in a construct where the CRE sequence was deleted. Furthermore, the mutants are less efficient than the wild type as templates for in vitro transcription elicited by liver nuclear extracts. Transcriptional activity decreases with the 20- and 28-bp insertions but is partially recovered with the 44-bp insertion. Partial purification of liver CRE- and CCAAT-binding proteins by high performance liquid chromatography on a Mono Q column and recombination of column fractions showed that a novel 73-kDa CRE-binding protein facilitates the association of the CCAAT-binding protein to the CCAAT site of the fibronectin gene.